**3. Effects of sulphonamides in the soil environment**

#### **3.1. Introduction to soil ecotoxicology**

*2.3.3. Influence of ionic strength*

668 Environmental Risk Assessment of Soil Contamination

in the literature.

**2.4. Available data on the presence of SAs in soils**

Another environmentally important factor that can affect SA sorption is ionic strength. But this has not been examined extensively. Ter Laak et al. [38] carried out sorption studies of SCP, among other compounds. Generally speaking, they did not observe any significant influence of ionic strength, except in the case of one soil (clay loam), in which sorption doubled when the CaCl2 concentration was raised from 0.006 to 0.2 M. The authors concluded that this increase in sorption was probably due to the neutral form of SCP increasing from 3.3 to 8.3% because of the decreasing pH. Protons are displaced from the cation-exchange sites by the addition of Ca2+ cations, which are ultimately responsible for the decrease in pH. Elevated cation concentrations near negatively charged soil surfaces, resulting in a decrease in the electrostatic repulsion of negatively charged sorbate molecules and soil particles, is another explanation considered by those authors. Srinivasan et al. [44] reported the different behaviour of SMX under conditions of increasing ionic strength. They explained the increasing *Kd* for SMX in the case of one soil as being due to cation bridging. Since positively charged calcium ions are present in the solution, bonding of anionic SMX to calcium is possible. In addition, the occurrence of a salting out effect, reducing the solubility of SMX in the salt solution so that it precipitates in the soil, was taken into consideration as a possible reason for the increase in sorption. The positive influence of ionic strength on sorption can also be attributed to the replacement of protons from the soil surface as the ionic strength increases, causing a slight reduction in pH, and shifting acidic SMX towards neutral forms that are more strongly sorbed than the anionic forms. Two other soils examined by Srinivasan et al. [44] exhibited an opposite and irregular trend in sorptive affinity of SMX, with elevated ionic strengths resulting in decreased sorption coefficients of SMX in the case of both soils. A slight decrease in sorption with increasing ionic strength of solute was also observed by Białk-Bielińska et al. [43] in the case of SDM and SGD and three soils. Srinivasan et al. [44] concluded that the ionic composition plays an important role in the sorption of ionizable organic compounds. Nevertheless, they, too, highlighted the necessity for further research in view of the conflicting results published

Although many methods have been developed in the past decade for the analysis of SAs in aqueous matrices, only a few are described in the literature for the determination of these contaminants in soil matrices. This is because the chemical analysis of pharmaceuticals from soil matrices is complicated by the need for extraction. Hence, our knowledge about the quantity of SAs in solid matrices is still limited. Nevertheless, the available literature data indicate their occurrence in agricultural soils after conventional fertilization. In a two-year monitoring study Höper et al. [78] determined SMZ at a concentration of 11 µg kg-1. Pawelzick et al. [79] reported a maximum concentration of 4.5 µg kg-1 for SMZ; these results are in agreement with Hu et al. [80], who demonstrated the occurrence of SMX (0.03 – 0.9 µg kg-1) and SCP (0.18 – 2.5 µg kg-1). Karcı et al. [81] found three SAs in agricultural soils in Turkey at concentrations even two orders of magnitude higher than those reported in previous studies: STZ (0.05 – 0.4 mg kg-1), SCP (0.05 – 0.1 mg kg-1) and SMX (0.05 – 0.1 mg kg-1). There are some SAs are commonly present in agricultural soils, though in fairly low concentrations (ppb and ppt levels), and are continuously being released into the environment via several routes (e.g. grazing animal faeces, manure spreading, WTP effluents). Such a state of affairs requires an Environmental Risk Assessment investigation, which should answer the basic question of whether the presence of SAs in soils poses a hazard. Ecotoxicology is the discipline that addresses this issue. It encompasses the study of organisms, populations, communities and ecosystems in terms of exposure to chemical agents, i.e. their transfer from the environment to organisms and their toxic effects. Simply put, it is the science of assessing the effects of toxic substances on ecosystems in order to protect these as a whole, rather than particular compart‐ ments, such as populations or organisms. In a practical manner, besides toxic effects, ecotox‐ icology explores the occurrence, distribution, accumulation and dissipation of anthropogenic toxic substances in ecosystems. The fundamental tools for this kind of research are ecotoxico‐ logical tests [82-83].

#### *3.1.1. Ecotoxicological tests*

These tests are a special group of quantitative research methods based on a thorough assess‐ ment of the impact of toxic substances (single or mixtures) on living organisms. Quantification of the results enables us to estimate the cumulative toxicity and the interactions between the introduced substances [82]. Obtaining such data enables scientists to extrapolate the results and define safe concentration levels in the ERA process (which is described in greater detail in Section 4 of this chapter).

In order to prepare valid ecotoxicity tests numerous factors need to be considered, the main ones being exposure time (acute or chronic), type of medium used, target species, toxic substance concentration range and choice of endpoint (e.g. mortality, growth inhibition, respiration).

Some 60 years ago scientists realized the need to establish uniform, standard test procedures in order to increase the repeatability and comparability of data obtained from tests. Researchers publish their own designs for tests together with results, enabling others to mimic the conditions for further experiments and allowing a better comparison of the results. Until now many standardized test procedures have been established by various environmental and governmental organizations/institutions. The best-known of these are the Organization for Economic Cooperation and Development (OECD), International Standards Organization (ISO), American Public Health Association (APHA), Environmental Protection Agency (EPA), American Society for Testing and Materials (ASTM) and International Seed Testing Associa‐ tion (ISTA), as well as many others from non-English speaking countries, like the the German Institute for Standardization (Deutsches Institut für Normung – DIN) and Polish Norms (Polskie Normy – PN). With this in mind it is common practice to perform tests strictly according to a chosen norm, or to modify just some aspects of a method as and when the conditions require this [82,84].

#### *3.1.2. Soil organisms in ecotoxicological studies*

The guidelines for ecotoxicological tests recommend using the best suited organisms. If the species stipulated in a guideline is unavailable, a similar one can be chosen, but it is important to select species that are extensively described in the literature. As species usually differ between ecosystems, their choice should take account of specific local conditions [82]. It is very important to realize that no single species is representative of all ecosystems; several singlespecies and multispecies tests have to be carried out in order to evaluate the behaviour of a toxic substance in an ecosystem.

Three main groups of organisms are evaluated in soil ecotoxicology: plants, microorganisms (microfauna) and animals (pedofauna). In the case of pedofauna, most ecotoxicological studies of soils are based on invertebrates and focus on worms, collembolans or enchytraeids because of their rapid reproduction times and easy maintenance. The most often examined endpoints here are weight change, survival, reproduction and behaviour (e.g. avoidance). Spermato‐ phytes are the most popular plants, in which the measured effects usually relate to physio‐ logical disorders, growth inhibition and seed germination. Microorganisms constitute a very sensitive indicator of chemical stress as there are many parameters that can be evaluated: the usual ones are respiration, nitrification and growth. However (as in the case of SAs), micro‐ organisms are also regularly evaluated for the occurrence and magnitude of increasing resistance towards pharmaceutical compounds [82-84].

#### **3.2. Available data on sulphonamide soil ecotoxicology**

An extensive literature review of data on SA soil toxicity has shown that there is a considerable gap in knowledge concerning the effects of these substances towards soil organisms. The vast majority of publications are dedicated to the analysis of microorganisms, followed by a small number of works on plants and just a few on pedofauna. Investigations involving the deter‐ mination of quantified dose-response data (such as EC50 – the median effective concentration) are rare. However, rather more experiments have been done to detect the ecotoxicity of SAs (e.g. effects observed at a single concentration). Several investigations into the accumulation of these drugs in plants and the problem of bacterial resistance have also been done and are summarized below.

### *3.2.1. Toxic effects of sulphonamides towards soil organisms*

substance concentration range and choice of endpoint (e.g. mortality, growth inhibition,

Some 60 years ago scientists realized the need to establish uniform, standard test procedures in order to increase the repeatability and comparability of data obtained from tests. Researchers publish their own designs for tests together with results, enabling others to mimic the conditions for further experiments and allowing a better comparison of the results. Until now many standardized test procedures have been established by various environmental and governmental organizations/institutions. The best-known of these are the Organization for Economic Cooperation and Development (OECD), International Standards Organization (ISO), American Public Health Association (APHA), Environmental Protection Agency (EPA), American Society for Testing and Materials (ASTM) and International Seed Testing Associa‐ tion (ISTA), as well as many others from non-English speaking countries, like the the German Institute for Standardization (Deutsches Institut für Normung – DIN) and Polish Norms (Polskie Normy – PN). With this in mind it is common practice to perform tests strictly according to a chosen norm, or to modify just some aspects of a method as and when the

The guidelines for ecotoxicological tests recommend using the best suited organisms. If the species stipulated in a guideline is unavailable, a similar one can be chosen, but it is important to select species that are extensively described in the literature. As species usually differ between ecosystems, their choice should take account of specific local conditions [82]. It is very important to realize that no single species is representative of all ecosystems; several singlespecies and multispecies tests have to be carried out in order to evaluate the behaviour of a

Three main groups of organisms are evaluated in soil ecotoxicology: plants, microorganisms (microfauna) and animals (pedofauna). In the case of pedofauna, most ecotoxicological studies of soils are based on invertebrates and focus on worms, collembolans or enchytraeids because of their rapid reproduction times and easy maintenance. The most often examined endpoints here are weight change, survival, reproduction and behaviour (e.g. avoidance). Spermato‐ phytes are the most popular plants, in which the measured effects usually relate to physio‐ logical disorders, growth inhibition and seed germination. Microorganisms constitute a very sensitive indicator of chemical stress as there are many parameters that can be evaluated: the usual ones are respiration, nitrification and growth. However (as in the case of SAs), micro‐ organisms are also regularly evaluated for the occurrence and magnitude of increasing

An extensive literature review of data on SA soil toxicity has shown that there is a considerable gap in knowledge concerning the effects of these substances towards soil organisms. The vast majority of publications are dedicated to the analysis of microorganisms, followed by a small number of works on plants and just a few on pedofauna. Investigations involving the deter‐ mination of quantified dose-response data (such as EC50 – the median effective concentration)

respiration).

conditions require this [82,84].

670 Environmental Risk Assessment of Soil Contamination

toxic substance in an ecosystem.

*3.1.2. Soil organisms in ecotoxicological studies*

resistance towards pharmaceutical compounds [82-84].

**3.2. Available data on sulphonamide soil ecotoxicology**

Literature results in the form of a dose-response relationship concerning soil organisms affected by SAs are relatively scant in comparison to the numbers available for aquatic organisms. Nonetheless, such material as has been gathered does allow us to establish some basic trends and consider the potential risk posed by SAs in the soil environment. Table 2 lists effective concentrations of SAs towards soil organisms determined for different taxonomic groups. Some of the endpoints deemed less relevant have not been included in Table 2, but they will nevertheless be mentioned in the following section.

To make the results presented in Section 3.2. more transparent, all cited concentrations have been recalculated into ppm units. However, it is of paramount importance to bear in mind that each individual study was designed separately; this implies, for example, differences between the media (soil or liquid) used. Careful thought is therefore advisable in this respect as one could grossly over- or underestimate the inferences drawn from the results. On that account, for a more detailed inquiry, we recommend that the reader refer to the original versions of the cited papers.

The pedofauna is the most understudied group of soil organisms mentioned in the literature: there have been just a handful of studies. Very interesting experiments using SMX, SDZ, SPY and SMZ were conducted on the nematode *Caenorhabditis elegans*, where several kinds of effects were evaluated (behavioural – movement, and growth – body length) during 24 – 96 hours. The second generation, not exposed to SAs, was examined in the same manner. The results showed that SAs affected growth and behaviour in all the exposed nematodes in a time- and concentration-dependent way. Also, as one might expect, behavioural effects were more sensitive than growth in all cases. Interestingly though, transgenerational effects were observed: the unexposed progeny of the examined nematodes exhibited significant toxic effects. This was speculated to correspond to the ability of SMX, SDZ, SPY to penetrate the placenta and the secretion of SDZ, SPY and SMZ in maternal milk [85-86]. A different study group evaluated the effects of SCP in a multispecies soil system, where one of the examined endpoints was the mortality of earthworms *Eisenia fetida*. In this case no effect was observed for up to 21 days of exposure to SCP at concentrations reaching 100 ppm [87].

Two independent groups performed studies of the potential impact of SMX and SMZ towards plant growth and soil quality. The plants investigated were rice *Oryza sativa* L., cucumber *Cucumis sativus* L., endive *Cichorium endivia* [88], lettuce *Lactuca sativa*, alfalfa *Medicago sativa* L. and carrot *Daucus carota* [89]. There are several differences between the approaches of the two groups, such as the time of exposure, range of concentrations or the types of tests used. Despite this, the results are comparable and some conclusions are shared. In all the investiga‐ tions SMX and SMZ were deemed to have the potential to affect soil organisms in environ‐ mentally relevant concentrations. Seed germination was found to be an insensitive endpoint. One of the groups [88] evaluated seed germination using the root length of seedlings in order to obtain better results. In nearly all cases SMX was found to be more effective than SMZ. Rice and carrot were found to be the most sensitive organisms with respective EC10 values of 0.1 ppm and 0.011 ppm [88-89]. Additionally, one group explored soil respiration and soil phosphatase activity: in these cases the respective EC10s for SMX were 7 ppm and 1 ppm [88]. The inference to be drawn here is that antibiotic residues in manure and soils may affect soil microbial and enzyme activities.

A relatively original investigation was performed to assess the impacts of anthropogenic stressors (i.a. SAs) on symbiotic plant-microbe relationships [90]. The authors studied the effects of SMX on the arbuscular mycorrhizal fungus *Glomus intraradices* grown on carrot *D. carota* root-organ cultures. The assay endpoints were root length (carrot), hyphae growth and spore production (fungus). The exposure period lasted up to 28 days and the highest concen‐ tration tested was 1 ppm. SMX was found to be effective at low concentrations towards both organisms: the respective EC50s for carrot and fungus (hyphae growth) were 0.0454 and 0.0451 ppm. Assessment of the endpoints was as follows: root lengths responded quickly to the presence of phytotoxic pharmaceuticals in the culture medium; hyphae length was a sensitive endpoint after 21 days' exposure; spore production required 28 days' exposure before significant differences could be detected [90].

The toxicity of STZ towards soybean (*Glycine max* (L.) Merr.) was evaluated as a potential nitrification inhibitor [91]. The effects were measured according to the growth of these plants. Fresh weight and dry weight of roots and plant tops were measured. The concentration range for STZ reached 200 ppm. STZ drastically reduced both main root elongation and lateral root development, the suppression increasing concentration-wise. Effects on soybean plants were detectable but statistically non-significant at a concentration of 10 ppm. STZ EC50 for dry root yield was equal to 29.5 ppm. It is worth noting that STZ inhibited root growth more than top growth.

The effects of sulphamonomethoxine sodium (SMM-Na) and sulphadiazine sodium (SDZ-Na) were investigated in three plant species: wheat *Triticum aestivum* L., Chinese cabbage *Brassica campestris* L. and tomato *Cyphomandra betacea* [92]. All of the plants exhibited linear correlations between the effects (root and shoot elongation) and SA concentrations. Seed germination was also considered, but was not sensitive to toxicity within the chosen range of SA concentrations. The tests were conducted over 2-5 days. The data acquired showed that wheat was the plant most sensitive to the toxicity of SDZ-Na with an IC50 = 28.1 ppm and that cabbage was the most sensitive to SMM-Na with an IC50 = 27.1 ppm. Worthy of note is the fact that in this study root and shoot elongation of the three crops exhibited different sensitivities, depending on the particular drug and plant species [92].

A study was developed specifically for SAs; it attempted to assess different susceptibility patterns of soil bacteria *Pantoea agglomerans* and standard antibiotic test bacteria *Pseudomonas aeruginosa*, depending on intercellular and environmental pH [93]. The results of the study revealed the effects at low concentrations (max. 20 ppm) of 8 SAs (SMX, STZ, SDZ, SDM, SMZ, SCP, SPY and SGD) at different pH values (from 5 to 8). The effects corresponding to the most sensitive pH values are listed in Table 2. The brief conclusion of this work is that the effects of SAs on microbial soil populations may depend closely on the ability of the bacteria to regulate their intercellular pH [93].


to obtain better results. In nearly all cases SMX was found to be more effective than SMZ. Rice and carrot were found to be the most sensitive organisms with respective EC10 values of 0.1 ppm and 0.011 ppm [88-89]. Additionally, one group explored soil respiration and soil phosphatase activity: in these cases the respective EC10s for SMX were 7 ppm and 1 ppm [88]. The inference to be drawn here is that antibiotic residues in manure and soils may affect soil

A relatively original investigation was performed to assess the impacts of anthropogenic stressors (i.a. SAs) on symbiotic plant-microbe relationships [90]. The authors studied the effects of SMX on the arbuscular mycorrhizal fungus *Glomus intraradices* grown on carrot *D. carota* root-organ cultures. The assay endpoints were root length (carrot), hyphae growth and spore production (fungus). The exposure period lasted up to 28 days and the highest concen‐ tration tested was 1 ppm. SMX was found to be effective at low concentrations towards both organisms: the respective EC50s for carrot and fungus (hyphae growth) were 0.0454 and 0.0451 ppm. Assessment of the endpoints was as follows: root lengths responded quickly to the presence of phytotoxic pharmaceuticals in the culture medium; hyphae length was a sensitive endpoint after 21 days' exposure; spore production required 28 days' exposure before

The toxicity of STZ towards soybean (*Glycine max* (L.) Merr.) was evaluated as a potential nitrification inhibitor [91]. The effects were measured according to the growth of these plants. Fresh weight and dry weight of roots and plant tops were measured. The concentration range for STZ reached 200 ppm. STZ drastically reduced both main root elongation and lateral root development, the suppression increasing concentration-wise. Effects on soybean plants were detectable but statistically non-significant at a concentration of 10 ppm. STZ EC50 for dry root yield was equal to 29.5 ppm. It is worth noting that STZ inhibited root growth more than top

The effects of sulphamonomethoxine sodium (SMM-Na) and sulphadiazine sodium (SDZ-Na) were investigated in three plant species: wheat *Triticum aestivum* L., Chinese cabbage *Brassica campestris* L. and tomato *Cyphomandra betacea* [92]. All of the plants exhibited linear correlations between the effects (root and shoot elongation) and SA concentrations. Seed germination was also considered, but was not sensitive to toxicity within the chosen range of SA concentrations. The tests were conducted over 2-5 days. The data acquired showed that wheat was the plant most sensitive to the toxicity of SDZ-Na with an IC50 = 28.1 ppm and that cabbage was the most sensitive to SMM-Na with an IC50 = 27.1 ppm. Worthy of note is the fact that in this study root and shoot elongation of the three crops exhibited different sensitivities, depending on the

A study was developed specifically for SAs; it attempted to assess different susceptibility patterns of soil bacteria *Pantoea agglomerans* and standard antibiotic test bacteria *Pseudomonas aeruginosa*, depending on intercellular and environmental pH [93]. The results of the study revealed the effects at low concentrations (max. 20 ppm) of 8 SAs (SMX, STZ, SDZ, SDM, SMZ, SCP, SPY and SGD) at different pH values (from 5 to 8). The effects corresponding to the most sensitive pH values are listed in Table 2. The brief conclusion of this work is that the effects of SAs on microbial soil populations may depend closely on the ability of the bacteria to regulate

microbial and enzyme activities.

672 Environmental Risk Assessment of Soil Contamination

significant differences could be detected [90].

particular drug and plant species [92].

their intercellular pH [93].

growth.



\* this author also examined the results for several other endpoints – see Section 3.2.1.

**Table 2.** Sulphonamide soil ecotoxicology – literature review

#### *3.2.2. Other relevant soil ecotoxicology data*

**Substance Type of test Species Critical effect Time Toxicity [ppm] Ref.**

bacteria *Pseudomonas*

bacteria *Pseudomonas*

wheat *Triticum aestivum*

tomato *Cyphomandra*

wheat *Triticum aestivum*

tomato *Cyphomandra*

bacteria *Pseudomonas*

*aeruginosa*

*aeruginosa*

*campestris* L.

*campestris* L.

*betacea*

*aeruginosa*

bacteria *Pantoea agglomerans*

rice *Oryza sativa* L.

cucumber *Cucumis*

rice *Oryza sativa* L.

cucumber *Cucumis*

endive *Cichorium endivia*

*sativus* L.

*sativus* L.

*betacea*

L.

L.

bacteria *Pantoea agglomerans*

bacteria *Pantoea agglomerans*

DIN 58959-7

674 Environmental Risk Assessment of Soil Contamination

**SDZ** DIN 58959-7

**SDZ-Na** OECD 1984

**SMM-Na** OECD 1984

**SDM** DIN 58959-7

ISTA 1985

OECD 1984 (modified)

**SDMD (SMZ)**

yield top

yield root

growth reduction – dry

growth reduction – dry

growth inhibition 24 h

growth inhibition 24 h

[92] cabbage *Brassica*

[92] cabbage *Brassica*

growth inhibition 24 h

root elongation inhibition \*

root elongation inhibition \*

root length (seed germination)

root length (plant growth)\*

tailored design soil microbe soil respiration 2 d EC50 = 13

ED50 = 33.5

ED50 = 29.5

EC50 = 5.47

EC50 = 0.77

EC50 = 2.85

EC50 = 0.5

2 d IC50 = 28.1

3 d IC50 = 31.3

5 d IC50 = 92.9

2 d IC50 = 120.7

3 d IC50 = 27.1

5 d IC50 = 88.0

4-5 d

20 d

EC50 > 20

EC50 = 2.05

EC50 = 45 NOEC = 1

EC50 > 300 NOEC = 1

EC50 = 37 NOEC = 0.1

EC50 = 43 NOEC = 1

EC50 > 300 NOEC = 100 [93]

[12]

[93]

[88]

Several publications by Migliore et al. have shed much light on the toxicity and bioaccumu‐ lation of SDM for different terrestrial plants [94-98]. The species included in the research belonged to two groups:


All the plants exhibited bioaccumulation and toxicity during post-germinative development at concentrations of 300 ppm, though of course to different extents. *Lythrum salicaria* L., exposed to lower concentrations, demonstrated a hormetic response. Crop plants accumulated SDM at dissimilar rates but always higher in roots than in foliage. In order to present the versatility of these results, those of additional research using other SAs and terrestrial plants are listed in Table 3.


All organisms in Table 3 belong to one of three groups: soil microorganisms **(sm)**, pedofauna **(p)** or terrestrial plants.

**Table 3.** Simplified list of published SA soil ecotoxicology research

As stated before soil microbiota is sensitive and easy to evaluate; hence, it is often exam‐ ined for several target effects. In some cases, however, the results are closely dependent on incubation time – OECD 209 guidelines recommend relatively short exposure times – which can lead to underestimated results. This issue has been mentioned by authors working with SAs [99-100]. Moreover, microorganisms are often not the primarily evalu‐ ated body in a test design. In fact effects on microbe communities are sought as addition‐ al results, helpful in monitoring the conditions in the test environment. Some research papers covering the soil ecotoxicity of SAs (not mentioned hitherto) are very briefly sum‐ marized in Table 3.

#### *3.2.3. Development of resistance of soil microorganisms to sulphonamides*

**Substance Organism Observed effects Ref.**

SDZ soil microorganisms respiration inhibition, adaptation [105] SDZ *Eisenia fetida* (p) no accumulation (uptake detected) [106] SDM *Rhizobium etli* (sm)–*Phaseolus vulgaris* L. symbiosis growth inhibition of both organisms [107]

12 SAs *Arthrobacter globiformis* (sm) no effect in t = 4 h [100] SMX *Salmonella typhimurium* (sm) mutagenic activity [108] SDZ *Zea mays* L. and soil microorganisms molecular-chemical pattern changes [109]

SDZ soil microorganisms soil community structure shift [111] SDZ *Triticum aestivum* L. accumulation (mainly in roots) [112]

SMX *Brassica campestris* L. minimal accumulation [115]

SDZ *Lactuca sativa*, *Daucus carota* not detected in subject plants [118]

All organisms in Table 3 belong to one of three groups: soil microorganisms **(sm)**, pedofauna **(p)** or terrestrial plants.

shifts

*Pisum sativum* L., *Cucumis sativus* L. accumulation (mainly in roots) [114]

accumulation, growth inhibition,

accumulation, toxic effects (root

non-extractable residues – low uptake by organisms [104]

accumulation, soil microbial biomass inhibition - higher effects for combined pollution

soil respiration and bacterial

root growth inhibition, necrotic changes, cytochrome c oxidase activity

accumulation [116]

accumulation [117]

community structure were influenced only after the addition of glucose

[70]

[110]

[113]

hormesis [94-98]

physiology impairment) [101-103]

*Panicum miliaceum* L., *Pisum sativum* L., *Zea mays* L., *Hordeum distichum* L., *Amaranthus retroflexus* L., *Plantago major* L., *Rumex acetosella* L. and *Lythrum*

*Salix fragilis* L., *Zea mays* L., *Hordeum vulgare* L.

*Brassica chinensis* L. and soil microorganisms

*Lupinus luteus*, *Pisum sativum* L.*, Lens esculenta* Medik., *Glycine max* (L.) Merr., *Vigna angularis*,

SMZ *Lolium perenne* L., *Poa pratensis* L., *Poa trivialis* L.,

*Solanum tuberosum* L., *Daucus carota*, *Zea mays* L.,

*Nasturtium officinale* R. Br.

*Lycopersicon esculentum* Mill.

**Table 3.** Simplified list of published SA soil ecotoxicology research

*Brassica rapa* L. and *Lumbricus terrestris* (p), activated

SDM

SDZ SDM SMZ

SDZ SMX

SDZ SMZ SMX

SMZ

SDM SMX

SDM SMR

*salicaria* L.

676 Environmental Risk Assessment of Soil Contamination

sludge (sm)

SDZ soil microorganisms

*Medicago sativa* L.

The current increasing interest in research into pharmaceutical residues in the environment has drawn the attention of scientists to the causation of bacterial resistance by antimicrobial residues. As the case of SA residues in terrestrial compartments is no exception, relevant research results have been published by several authors.

The effect of SCP together with pig slurry was examined during a three-week exposure, using Biolog® multiwall plates to determine pollution-induced community tolerance (PICT). Several physiological processes were monitored as well as community-level physiological profiling (CLPP) [119]. As a result of the tests it was established that the soil microbial community's tolerance increased as soon as 7 days following exposure. Indeed, a SCP concentration of 7 ppm was sufficient to trigger the first effects. An increase in tolerance has been reported for a procedure comparable to normal agricultural practice [119].

Several investigations have been conducted with SDZ in manure. It was established that such a combination synergistically increased antibiotic resistance in bacteria. Some of the explored variations of the tests included the use of a multispecies system (microcosm) with soil bacteria, the preparation of tests on different kinds of soil and multiple amend‐ ment with pig manure. SA resistance genes were detected using hybridization and the polymerase chain reaction (PCR). In all cases there was a significant increase in resistance, though differing in extent depending on the test design. Following amendment, the bacte‐ rial populations carrying the SA resistance genes introduced to the soil declined strongly in the first weeks; nonetheless, they have the potential to be present for several months [120-122].

Sulphamethoxazole (SMX) was also examined with respect to its effect on soil bacteria. Two methods were used to assess PICT: leucine incorporation and Biolog® plates. Community structure was assessed using phospholipid fatty acid (PLFA) analysis, CLPP and bacterial growth. No effect was seen at 1 ppm SMX. At higher concentrations (20 – 500 ppm) effects were significant but relatively small (a ca twofold increase in community tolerance). None‐ theless, the impact of SMX on soil reflected both the direct inhibition of bacterial growth rates and changes in community structure [123].
