THE EFFECTS OF SELECTED ANTIBIOTICS ON NITROGEN UPTAKE BY Spirodela punctata
By
Cory M. Jones
A Thesis
Presented to
The Faculty of Humboldt State University
In Partial Fulfillment
Of the Requirements for the Degree
Master of Science
In Natural Resources: Wastewater Utilization Program
February, 2010
ABSTRACT
The Effects of Selected Antibiotics on Nitrogen Uptake by Spirodela punctata
Cory M. Jones
The purpose of this study was to determine the effects of nitrogenous compound
removal by the aquatic macrophyte, Spirodela punctata, when exposed to three selected
antibiotics. Recent research has shown that certain antibiotics target the chloroplasts of
aquatic species such as Lemna and Myriophyllum. Studies have demonstrated antibiotic
toxicity to Lemna gibba at concentrations as low as 10 µg/L. Meanwhile, antibiotic concentrations in domestic wastewater lie in the nanogram to microgram range with an average of approximately 50 µg/L.
In this study, Spirodela punctata was grown in a mineral salts medium containing the antibiotics chlortetracycline, lomefloxacin, and sulfamethoxazole in concentrations ranging from 10 µg/L to 300 µg/L. Fronds were allowed to grow in the medium for seven, fourteen, and twenty-one day periods. Following the growth periods, the medium was analyzed for nitrate and total nitrogen concentrations. Dry weights of fronds were taken and the dried plant material was analyzed for Total Kjeldahl Nitrogen (TKN) content.
Effective concentrations (EC25 and EC50) that impacted total nitrogen and nitrate removal from the growth medium as well as dry weights and Total Kjeldahl Nitrogen of
the plant tissue were calculated. Of the antibiotics tested, chlortetracycline had the most
iii
significant responses. The antibiotic reduced nitrate and total nitrogen removal from the medium and decreased plant biomass. For nitrate removal chlortetracycline had an EC25 of 32 µg/L after three weeks of exposure. Dry weight EC25 for chlortetracycline was 73
µg/L after seven days of exposure to the antibiotic. Control treatments containing the antibiotic and no plant material had similar results indicating that the compounds added nitrogen.
Conversely, other compounds tested resulted in very few significant responses.
Lomefloxacin only showed significance for nitrate removal during week two and total nitrogen for week one. Surprisingly, the antibiotic seemed to stimulate the ability of
Spirodela punctata to remove total nitrogen during the first week of exposure.
Sulfamethoxazole had only one significant response during the test period. The combination of the three antibiotics resulted in only a reduction in nitrate removal during the first week after which there was no significant effect.
Overall, selected antibiotics had little direct effect on ability of Spirodela punctata to remove nitrogenous compounds. However, with respect to chlortetracycline, the reduction in the production of biomass resulted in the reduction in nitrate and total nitrogen removal. This reduction in biomass is perhaps the greatest impact on S. punctata’s ability to remove nitrogenous compounds from wastewater.
iv
ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincere appreciation towards Dr.
Kristine Brenneman for the commitment, support, and expertise that she lent me
throughout my graduate school experience. Dr. Brenneman was extremely patient with
me through multiple thesis revisions and motivated me to keep working. I could not have
done it without her. Secondly, I would like to thank Dr. Bill Bigg and his wife, Donna, for their help with statistics, experimental design, and the Total Kjeldahl Nitrogen analysis. They will be missed at Humboldt State University. Thanks to Ryan Faria-Cecil
for all of his help during those long hours in the laboratory. I would also like to express
my gratitude towards the city of Arcata for funding my research. My committee
members, Dr. Frank Shaughnessy and Dr. Margaret Lang are greatly appreciated for their
efforts providing insight regarding my project and for time spent reviewing my thesis.
Thanks to the biology department for lending me laboratory materials without which I
wouldn’t have been able to perform my research. Finally, I would like to thank my
family for all of the support, financially and otherwise, that allowed me to achieve my
goal. I am especially indebted to my beautiful wife, Lynette, who always supported and
believed in me throughout this entire project.
v
TABLE OF CONTENTS
Page
ABSTRACT...... iii
ACKNOWLEDGEMENTS...... v
TABLE OF CONTENTS...... vi
LIST OF TABLES...... vii
LIST OF FIGURES...... viii
INTRODUCTION...... 1
MATERIALS AND METHODS...... 5
Response Variables and Statistics...... 7
RESULTS...... 8
DISCUSSION...... 21
LITERATURE CITED...... 26
PERSONAL COMMUNICATION CITED...... 29
APPENDIX A...... 30
Antibiotic Molecular Structures...... 30
vi
LIST OF TABLES
Table Page
1 The p-values for significant responses for indicated assays of growth media treated with antibiotics as determined using analysis of variance (ANOVA) in the general linear model...... 9
2 The p-values and lowest-observable-effect concentrations (LOECs) for indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using analysis of variance (ANOVA) in the general linear model and Dunnett’s two-sided test for treatment-control comparison...... 11
3 Effective concentrations that cause a 25% change from the control for the indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using curve fitting modeling techniques at the 95% confidence interval...... 12
4 Effective concentrations that cause a 50% impact on the indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using curve fitting modeling techniques at the 95% confidence interval...... 13
vii
LIST OF FIGURES
Figure Page
1 Significant (p<0.05) effects of the antibiotic chlortetracycline on nitrate removal by Spirodela punctata...... 14
2 Significant (p<0.05) effects of the antibiotic chlortetracycline on total nitrogen removal by Spirodela punctata...... 15
3 Significant (p<0.05) effects of the antibiotic chlortetracycline on frond dry weight and Total Kjeldahl Nitrogen percentage of Spirodela punctata...... 16
4 Significant (p<0.05) effects of the antibiotic lomefloxacin on nitrate removal and total nitrogen removal by Spirodela punctata...... 18
5 The significant (p<0.05) effect of the sulfonamide antibiotic sulfamethoxazole on total nitrogen removal by Spirodela punctata...... 19
6 The significant (p<0.05) effect of the combination of three antibiotics (chlortetracycline, lomefloxacin, and sulfamethoxazole) on nitrate removal by Spirodela punctata...... 20
viii
INTRODUCTION
Recent studies have provided evidence that antibiotics have phytotoxic effects on
vascular plants including Lemna and Myriophyllum species (Brain et al. 2003, Brain et al.
2004, Pomati et al. 2004). The primary focus of these studies has been on the
morphological effects that antibiotics impose including reduction of chlorophyll and
biomass. To my knowledge, nothing is known about the effect antibiotics have on
nutrient removal by such aquatic species.
Lemnaceae have long been shown to be effective in wastewater treatment (Harvey
and Fox 1973, Sutton and Ornes 1975, Hillman and Culley 1978). Lemnaceae are
efficient in reducing nitrogen and phosphorus levels from various polluted water sources
including animal wastes and municipal wastewater. A study by Tripathi (20003) showed
that Lemna minor removed 62.5% of total nitrogen and 58.8% of phosphorus from
secondarily treated dairy effluent in eight-weeks. Furthermore, Cheng et al. (2001)
reported that within a 20-day period Spirodela punctata removed all NH4-N and P04-P
from swine lagoon water. Initial nutrient concentrations were high, containing 240.0
mg/l and 31.0 mg/l for ammonium and phosphate, respectively. Results indicated that S.
punctata performed well in water containing high nitrogen and phosphate conditions. In
addition, Lemnaceae are effective in reducing total solids and chemical oxygen demand
(COD). For example, Mandi (1994) showed that urban wastewater treated with Lemna
gibba removed total suspended solids (TSS) and chemical oxygen demand (COD) by
61% and 82%, respectively
1 2 Antibiotics have received increasing attention due to their negative effect on wastewater treatment (Huang et al. 2001). Sewage contains pharmaceutical compounds via domestic, hospital, and animal husbandry operational wastes (Halling-Sorensen et al.
1998). Antibiotics are not entirely metabolized when used to treat disease; thus, much is excreted through urine and feces (Pomati et al. 2004). It is estimated as much as 70 to
80% of antimicrobial pharmaceuticals are not metabolized and enter the environment in a deleterious form (Samuelsen et al. 1992). Once in the wastewater treatment system, antibiotics reduce bacterial communities (e.g. nitrifying bacteria) necessary for effective treatment of wastewater (Jensen 2001). Alternatively, antibiotics are responsible for resistant strains of disease causing bacteria. Studies have shown that resistant forms of
Salmonella typimurium, Escherichia coli and Enterococcus sp. increased significantly as use of antibiotics in farming increased (Jones et al. 2001). Furthermore, antibiotics, such as oxytetracycline, are known to be endocrine disrupters (Flemming et al. 2003). In addition to the numerous synthetically produced antibiotics, both prokaryotic and eukaryotic organisms naturally produce antimicrobial peptides and proteins. Eukaryote representatives include vertebrates, invertebrates, plants, and fungi (Marx 2004).
Typical concentrations of individual antibiotics in untreated wastewater are relatively low, most being in the nanogram per liter to microgram per liter range. Average antibiotic concentration within urban wastewater is approximately 50 µg/L (Huang et al. 2001,
Kummerer 2001); however, the combined concentrations of pharmaceuticals being released can have a significant impact (Jones et al. 2001). Also, most antibiotics have
3 relatively short half-lives but continual addition to the environment gives them a ‘pseudo- persistent’ nature (Daughton and Ternes 1999).
Antibiotics are also known to interfere with photosynthesis. Chloroplasts evolved when a photosynthetic cyanobacterium was phagocytized and retained within a unicellular eukaryote (Margulis 1981). Although the size of the cyanobacterial genome within the chloroplast has become reduced over time, it is still mostly prokaryotic and therefore is vulnerable to antibiotic compounds. Due to similarities between bacterial and chloroplast genomes, mechanisms involved in DNA replication, transcription, and translation are affected (McFadden and Roos 1999). Ebringer (1972) demonstrated that of 144 antibiotics studied, 46 caused irreversible damage to plastids and that most inhibited synthesis of chlorophyll in the alga Euglena gracilis. Loss of chloroplasts and decreased chlorophyll production resulted in what is known as a “bleaching effect”. The study concluded that only those antibiotics that inhibit DNA synthesis or protein synthesis caused the bleaching effect in E. gracilis.
Although studies on the effect of antibiotics on algae have been done, little is known about the impact to higher plants. A study on the effects of 25 antibiotics to the duckweed, Lemna gibba, demonstrated phytotoxicity to certain antibiotics at concentrations as low as 10 µg/L (Brain et al. 2003). Of the three classes of antibiotics studied, including fluoroquinolones, sulfonamides, and tetracyclines, based on chlorophyll content, frond production, and wet weight the most phytotoxic were lomefloxacin, sulfamethoxazole, and chlortetracycline respectively. Wet weight EC25
4 values were found to be lowest in sulfamethoxazole at 37 µg/L, followed by lomefloxacin at 38 µg/L, and finally chlortetracycline at 114 µg/L.
The purpose of my study was to determine the effect of three phytotoxic antibiotics on the ability of Spirodela punctata to remove nitrogenous compounds from wastewater.
Spirodela punctata is a free-floating aquatic macrophyte in the Lemnaceae family. This species of duckweed was chosen because it is commonly found in California wetlands and constructed wastewater treatment wetlands. It is also effective in removal of nitrogenous compounds found in wastewater (Cheng et al. 2001).
MATERIALS AND METHODS
Spirodela punctata was obtained from the Humboldt State University greenhouse.
Dr. Michael Mesler provided species confirmation. (Spirodela punctata will be renamed
Landoltia punctata in the forthcoming Jepson Manual, personal communication Mesler,
2006).
Plants were sterilized by asceptically submerging individual fronds in a test tube with 20% sodium hypochlorite solution (Bowker et al. 1980). Each tube was agitated until frond margins became chlorotic. Fronds were removed immediately and at 30- second intervals thereafter until completely chlorotic. After bleaching, viable fronds were rinsed in sterile distilled water. Plants were transferred to test tubes containing 10 ml sterile Hoagland’s + 1% sucrose medium with pH adjusted to 6.0 using 0.1 N sodium hydroxide. Fronds were grown for a period of seven days under 40 watt fluorescent lights. Sterility was determined by streaking an aliquot of medium onto Tryptic Soy
Agar (Difco, St. Louis, MO) and incubating at room temperature for five days.
Sterile fronds were aseptically transferred to covered Pyrex® 250 mL Erlenmeyer flasks containing 200 mL of sterile Hoagland’s medium. These vessels were placed under 40-watt fluorescent light and allowed to grow for a period of fourteen days. These fronds became sterile stock cultures.
For each assay, twelve 250 ml Erlenmeyer flasks were filled with 90 mL of sterile
Hoagland’s + 1% sucrose medium. For each antibiotic, concentrations of 10 µg/L, 30
µg/L, 100 µg/L, and 300 µg/L were prepared. The three antibiotics used in the assay
5 6 were chlortetracycline, lomefloxacin, and sulfamethoxazole. Antibiotic solutions were asceptically added to flasks using a 5 cc or 1 cc syringe of Fisher Brand® ultracleaning, mixed esters of cellulose 0.45µm filter units (Fisher Scientific, Pittsburgh, PA) bringing total volumes to 100 mL per flask. Additions of antibiotics were performed in a laminar- flow hood using a “stir and drip” method. Thirty sterile fronds of Spirodela punctata were transferred to the twelve 250 ml flasks. Ten milliliters of sterile distilled water and
30 sterile fronds served as the control. Furthermore, nine separate flasks containing no plants were prepared with each antibiotic at each concentration plus one with 10 mL of sterile distilled water to determine if the antibiotics have an effect on nitrogen content.
The assay was performed in triplicate (n = 78).
In addition to plants exposed to individual antibiotics, sterilized fronds were exposed to equal combinations of the three antibiotics at final concentrations of 10 µg/L, 30 µg/L,
100 µg/L, and 300 µg/L. Ten milliliters of sterile distilled water and 30 sterile fronds served as the control. Furthermore, four flasks containing the combined antibiotics and one with sterile distilled water were prepared with no plants to determine if the antibiotics have an effect on nitrogen content. The assay was performed in triplicate (n = 30).
Two 40-watt fluorescent bulbs provided light for optimal plant growth (5350-5800 lux). Ballasts were set 30 cm above plant cultures. All samples were exposed to the lights for 24 hours a day according to standard procedure (American Society of Testing
Materials, 1998). The procedure was repeated for 7, 14, and 21-day growth periods.
Following growth periods, the medium in all flasks was tested for nitrate and total nitrogen concentrations. Nitrate concentrations were determined using the HACH Nitrate
7
Mid-Range (0 to 5.0 mg/L NO3-N) Method #8171. Total nitrogen concentration was determined using the HACH Total Nitrogen HR Test ‘N Tube™ (10-150 mg/L N) TNT
Persulfate Method #10072. Samples were read using a HACH DR 890 Colorimeter®
(HACH Company, Loveland, CO).
Plants were removed after incubation and placed in a drying oven set at 80º C for 24 hours. The dried plant tissue was assayed for total nitrogen content using a Total
Kjeldahl Nitrogen acid digest procedure. The digest was performed using a Lachat BD-
46 Block Digester® (Lachat Instruments, Loveland, CO). Following digestion, the samples were read using a Bausch & Lomb Spectronic 101® Spectrophotometer (Bausch
& Lomb, Rochester, NY) against a standard curve.
Response Variables and Statistics
Response variables for the plant tissue were dry weight and Total Kjeldahl Nitrogen concentrations. Variables for the medium were nitrate and total nitrogen concentrations.
A one-way analysis of variance (ANOVA) was performed on each variable for each antibiotic or combination of antibiotics at each concentration using NCSS 2000 Statistical
System for Windows. Analyses were conducted to compare means between the single independent variables. Those response variables identified as significant (p < 0.05), were analyzed using Dunnett’s two-sided test for treatment-control comparison to determine lowest-observable-effect concentrations (LOEC). Furthermore, significant variables were analyzed using curve-fitting modeling techniques in NCSS to determine effective concentration.
RESULTS
All three antibiotics in this study, in addition to the combination of the three, added a significant amount (p < 0.05) of nitrogenous compounds, including nitrate, to the
growth medium at various times over the three-week period. However, no antibiotics
added more than 0.95 mg/L of nitrate or 4.30 mg/L of total nitrogen to the medium
(Table 1).
Additions of different antibiotic compounds to plant samples resulted in different morphological effects to fronds of Spirodela punctata. Chlortetracycline imposed the
bleaching effect on new fronds within four days especially at higher concentrations (30-
300 µg/L). New fronds exhibited a light yellowing throughout until approximately day-
ten when they began to take on a green color. Those samples treated with
sulfamethoxazole developed new fronds with near-white margins particularly at the
highest concentrations (100 and 300 µg/L). These fronds initially affected by
sulfamethoxazole remained bleached at the margins until the end of the three-week
period. Any new fronds developing later in the growth period photosynthesized
normally; thus, producing a green color. The combination of the three antibiotic
compounds resulted in morphological effects similar to that of chlortetracycline alone.
Following three days after exposure to the compounds, new fronds exhibited the
bleaching effect, especially at higher concentrations (30-300 µg/L). However, unlike
chlortetracycline treated fronds, those treated with the combination of antibiotics
exhibited chlorosis for only two days at which time they developed a green color.
8
Table 1. The p-values for significant responses for indicated assays of growth media treated with antibiotics as determined using analysis of variance (ANOVA) in the general linear model (NCSS).a
Compound Week Nitrate Total nitrogen
Chlortetracycline 1 0.0178 0.1315
2 0.1303 0.1466
3 0.0030 0.9738
Lomefloxacin 1 0.8210 0.0895
2 0.0098 0.1488
3 0.0012 0.1454
Sulfamethoxazole 1 0.0118 0.0011
2 0.0824 0.0226
3 0.0951 0.0026
Combination 1 0.5246 0.3348
2 0.0056 0.1446
3 0.0239 0.1636 a significant p-values in bold 9
10 Lomefloxacin was the only compound tested that did not impose any noticeable
morphological effects on samples of Spirodela punctata.
Plant samples exposed to growth medium containing the tetracycline antibiotic
chlortetracycline had the most statistically significant responses for each endpoint per
week of exposure compared to other compounds tested. Results obtained from weeks
one and three showed significance in remaining nitrate concentrations in the growth
medium (p<0.05). Both weeks had LOEC values of 30 µg/L chlortetracycline.
Additionally, total nitrogen remaining in the medium was greater in the antibiotic treatments compared to the control during all three weeks of sample exposure to chlortetracycline, resulting in LOECs of 30 µg/L for each. Mean dry weights for weeks one and two resulted in LOEC values of 30 µg/L. Finally, Total Kjeldahl Nitrogen present in plant tissue exposed to chlortetracycline was less than the control following the third week of exposure, with a LOEC value of 100 µg/L (Table 2). The EC25 and EC50
values for plant samples treated with chlortetracycline ranged from 32 µg/L to 290 µg/L
for samples that were found to be significant in the ANOVA analysis (Tables 3, 4;
Figures 1, 2, 3).
The fluoroquinolone antibiotic, lomefloxacin, resulted in only two significant
responses during three weeks of exposure. During the first week of exposure,
lomefloxacin had an effect on total nitrogen removal (Table 2). The LOEC for total
nitrogen was 10 µg/L, the EC25 value was 7 µg/L, and the EC50 was 8 µg/L (Tables 2, 3,
4). During this first week, Spirodela punctata removed more total nitrogen in the
medium at higher antibiotic concentrations than the
11 Table 2. The p-values and lowest-observable-effect concentrations (LOECs) for indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using analysis of variance (ANOVA) in the general linear model and Dunnett’s two-sided test for treatment-control comparison (NCSS).
Nitrate Total nitrogen Dry weight Total Kjeldahl nitrogen Compound Week p-Value LOEC p-Value LOEC p-Value LOEC p-Value LOEC
Chlortetracycline 1 <0.0001 30 0.0152 30 0.0002 30 0.1101 NSD
2 0.0939 NSD 0.0093 30 0.0010 30 0.0983 NSD
3 0.0027 30 0.0461 30 0.0865 NSD 0.0167 100
Lomefloxacin 1 0.0998 NSD 0.0156 10 0.3359 NSD 0.0528 NSD
2 0.0137 30 0.5875 NSD 0.2095 NSD 0.7509 NSD
3 0.0501 NSD 0.4672 NSD 0.4423 NSD 0.2509 NSD
Sulfamethoxazole 1 0.5231 NSD 0.2478 NSD 0.2750 NSD 0.6148 NSD
2 0.0529 NSD 0.0228 10 0.0780 NSD 0.3102 NSD
3 0.4987 NSD 0.0598 NSD 0.5585 NSD 0.2277 NSD
Combination 1 0.0028 10 0.9311 NSD 0.9387 NSD 0.5198 NSD
2 0.0808 NSD 0.7248 NSD 0.5938 NSD 0.7272 NSD
NSD = no significant difference, indicating no significance between treatment means and consequently LOEC was not calculated. LOECs are listed in µg/L. Significant (<0.05) responses are in bold.
11
12 Table 3. Effective concentrations that cause a 25% change from the control for the indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using curve fitting modeling techniques at the 95% confidence interval.a
Nitrate Total nitrogen Dry weight Total Kjeldahl nitrogen 2 2 2 2 Compound Week EC25 r EC25 r EC25 r EC25 r
Chlortetracycline 1 134 0.81 77 0.58 73 0.98 >300 NC
2 >300 NC 100 0.27 113 0.77 >300 NC
3 32 0.92 89 0.83 >300 NC 127 0.30
Lomefloxacin 1 >300 NC 7 0.52 >300 NC >300 NC
2 60 0.62 >300 NC >300 NC >300 NC
3 >300 NC >300 NC >300 NC >300 NC
Sulfamethoxazole 1 >300 NC >300 NC >300 NC >300 NC
2 >300 NC 160 0.30 >300 NC >300 NC
3 >300 NC >300 NC >300 NC >300 NC
Combination 1 44 0.93 >300 NC >300 NC >300 NC
2 >300 NC >300 NC >300 NC >300 NC
3 >300 NC >300 NC >300 NC >300 NC a EC25 values are reported in µg/L of the respective compound. NC = not calculated due to a lack of significance between treatment means in ANOVA analysis.
12
13 Table 4. Effective concentrations that cause a 50% impact on the indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using curve fitting modeling techniques at the 95% confidence interval.a
Nitrate Total nitrogen Dry weight Total Kjeldahl Nitrogen 2 2 2 2 Compound Week EC50 r EC50 r EC50 r EC50 r
Chlortetracycline 1 290 0.81 103 0.58 138 0.98 >300 NC
2 >300 NC 133 0.27 156 0.77 >300 NC
3 46 0.92 277 0.83 >300 NC 210 0.30
Lomefloxacin 1 >300 NC 8 0.52 >300 NC >300 NC
2 68 0.62 >300 NC >300 NC >300 NC
3 >300 NC >300 NC >300 NC >300 NC
Sulfamethoxazole 1 >300 NC >300 NC >300 NC >300 NC
2 >300 NC 161 0.30 >300 NC >300 NC
3 >300 NC >300 NC >300 NC >300 NC
Combination 1 58 0.93 >300 NC >300 NC >300 NC
2 >300 NC >300 NC >300 NC >300 NC
3 >300 NC >300 NC >300 NC >300 NC
a EC50 values are reported in µg/L of the respective compound. NC = not calculated due to a lack of significance between treatment means in ANOVA analysis.
13
1414
Chlortetracycline Week #1 Nitrate
105
control 10 ug/L 85 30 ug/L 100 ug/L
300 ug/L
65 antibiotic concentration
Chlortetracycline Week #3 Nitrate
6
5
4 control 10 ug/L 3 30 ug/L 100 ug/L 2 300 ug/L
1
0 antibiotic concentration
Figure 1. Significant (p<0.05) effects of the antibiotic chlortetracycline on nitrate removal by Spirodela punctata. Error bars represent standard error of the means of three replicates.
15
Chlortetracycline Week #1 Total Nitrogen
95
control 10 ug/L 85 30 ug/L 100 ug/L
300 ug/L
75 antibiotic concentration
Chlortetracycline Week #2 Total Nitrogen
50
control
46 10 ug/L 30 ug/L 100 ug/L
42 300 ug/L
38 antibiotic concentration
Chlortetracyline Week #3 Total Nitrogen
20
control 10 ug/L 10 30 ug/L 100 ug/L
300 ug/L
0 antibiotic concentration
Figure 2. Significant (p<0.05) effects of the antibiotic chlortetracycline on total nitrogen removal by Spirodela punctata. Error bars represent standard error of the means of three replicates.
16
Chlortetracycline Week #1 Dry Weight
0.04
0.03 control 10 ug/L 0.02 30 ug/L
100 ug/L 300 ug/L 0.01
0 antibiotic concentration
Chlortetracycline Week #2 Dry Weight
0.1
0.09
control 0.08 10 ug/L 30 ug/L 100 ug/L
0.07 300 ug/L
0.06
0.05 antibiotic concentration
Chlortetracycline Week #3 Total Kjeldahl Nitrogen
3.75
3.25 control 10 ug/L 30 ug/L 100 ug/L 2.75
300 ug/L
2.25 antibiotic concentration
Figure 3. Significant (p<0.05) effects of the antibiotic chlortetracycline on frond dry weights and Total Kjeldahl Nitrogen percentage of Spirodela punctata. Error bars represent standard error of the means of three replicates.
17 control (Figure 4). During week two there was a significant removal of nitrate (p<0.05)
(Table 2). The LOEC for this endpoint was 30 µg/L, the EC25 value was 60 µg/L, and the
EC50 was 68 µg/L (Tables 2, 3, 4; Figure 4).
The addition of the sulfonamide antibiotic sulfamethoxazole resulted in only one significant (p<0.05) response during all three weeks of exposure. The response variable total nitrogen for week two had a LOEC value of 10 µg/L, an EC25 of 160 µg/L, and an
EC50 of 161 µg/L (Tables 2, 3, 4). This sample resulted in Spirodela punctata removing more total nitrogen in the medium at higher antibiotic concentrations than the control
(Figure 5).
Plant samples exposed to growth medium containing a combination of the three antibiotics resulted in only one significant (p<0.05) response during all three weeks of the study. Week one was significant with respect to decreased nitrate removal, resulting in a
LOEC value of 10 µg/L, an EC25 of 44 µg/L, and an EC50 of 58 µg/L (Tables 2, 3, 4;
Figure 6).
18
Lomefloxacin Week #1 Total Nitrogen
80
75 Control 10 ug/L 30 ug/L 100 ug/L 70 300 ug/L
65 Antibiotic Concentration
Lomefloxacin Week #2 Nitrate
90
80 Control 10 ug/L 70 30 ug/L
100 ug/L 300 ug/L 60
50 Antibiotic Concentration
Figure 4. Significant (p<0.05) effects of the antibiotic lomefloxacin on nitrate removal and total nitrogen removal by Spirodela punctata. Error bars represent standard error of the means of three replicates.
19
Sulfamethoxazole Week #2 Total Nitrogen
60
50 Control 10 ug/L 40 30 ug/L 100 ug/L
300 ug/L 30
20 Antibiotic Concentration
Figure 5. The significant (p<0.05) effect of the sulfonamide antibiotic sulfamethoxazole on total nitrogen removal by Spirodela punctata. Error bars represent the standard error of the means of three replicates.
20
Combination Week #1 Nitrate
90
80 Control 10 ug/L 30 ug/L
100 ug/L 70 300 ug/L
60 Antibiotic Concentration
Figure 6. The significant (p<0.05) effect of the combination of three antibiotics (chlortetracycline, lomefloxacin, and sulfamethoxazole) on nitrate removal by Spirodela punctata. Error bars represent the standard error of the means of three replicates.
21 DISCUSSION
This study was performed to determine the effects of selected antibiotics on nitrogen removal by Spirodela punctata. Particularly, in respect to wastewater treatment, the intention was to better understand the impacts that emerging contaminants, such as antibiotics, have on aquatic plants residing in wastewater treatment wetlands. Effects were quantified by calculating LOEC, EC25, and EC50 values of antibiotic concentrations for samples determined to be significant by analysis of variance. The tetracycline antibiotic chlortetracycline (Appendix A) resulted in more impacts on dry weights, total nitrogen and nitrate removal, and TKN present in the plant tissue compared to all other treatments combined.
The addition of antibiotic compounds to the medium containing no plants resulted in significant increases in nitrate concentrations. Therefore, some impacts on the plant treatments can be directly attributed to the addition of nitrogen by the antibiotics themselves. For example, consider the assay of nitrate as nitrogen in the growth medium for the first week of chlortetracycline exposure in the medium and to plants (Figure 1).
Significance was found in both (Tables 1 and 2). In fact, there are similarities in week three nitrate concentrations in the samples with and without plants. Consequently, the nitrate concentration present in the medium is a direct result of the antibiotics themselves.
Lower values present in plant samples are assumed to be the result of nutrient uptake by the duckweed fronds. These results indicate that antibiotic compounds such as chlortetracycline are potential sources of nitrates in wastewater treatment systems and
22 have an effect on nutrient loading in addition to damaging the plants ability to remove nitrogen from the water.
There were no similarities for total nitrogen concentrations between plant and
non-plant treatments. Total nitrogen values are significantly higher at increased
antibiotic concentrations during all three weeks of S. punctata frond exposure to
chlortetracycline (Table 2). No significant difference was found with respect to total
nitrogen in samples containing medium alone (Table 1). The total nitrogen
concentrations increased with greater amounts of chlortetracycline during each week of
the three-week period. This indicates that the plants were inhibited by the antibiotic and
removal of total nitrogen decreased as the concentration of chlortetracycline increased.
Furthermore, decreases in plant dry weight occurred at higher concentrations of the
antibiotic (Tables 2, 3, 4). In other words, less plant biomass resulted in less removal of
nutrients. This is thought to be due to the protein synthesis targeted by tetracycline group
antibiotics. The inhibition of protein synthesis resulted in decreased biomass that, in turn,
resulted in less removal of nitrogen required for cellular processes.
Total nitrogen includes organic nitrogen, nitrate, nitrite and ammonia. Although
chlortetracycline resulted in the addition of a significant amount of nitrate to the medium,
the impact it had on plants may have been a diminished ability to remove other forms of
nitrogen when antibiotic concentrations were increased. Chlortetracycline, after three
weeks of exposure, was the only compound tested that had a significant effect on Total
Kjeldahl Nitrogen (TKN) concentrations in the tissue of Spirodela punctata (Table 3).
The correlation coefficient (r2) for week three TKN values was 0.30, indicating poor
23 correlation. On the other hand, unlike total nitrogen, TKN only includes organic forms of
nitrogen and ammonia. Therefore, although S. punctata contains the enzyme nitrate reductase, reducing nitrate to nitrite, it is possible that other forms of inorganic nitrogen were not detected in the TKN analysis that may have indicated significance (Lillo et al.,
1998).
Similar to chlortetracycline, addition of lomefloxacin resulted in significant increases in nitrate concentrations when control treatments were present (Table 1).
Following weeks two and three, nitrate concentrations were significantly higher at increasing concentrations of the antibiotic indicating the antibiotic contributed nitrates to
the medium. Although lomefloxacin contributed nitrates to treatments without plants,
treatments containing S. punctata fronds from week one performed better at removing
total nitrogen at higher concentrations of the antibiotic than control treatments (Figure 2).
It is possible lomefloxacin had a stimulatory effect on the ability of the plants to remove
nitrogen.
Significant effects of lomefloxacin in S. punctata samples were also found in
week two nitrate concentrations (Figure 2). Higher nitrate values occurred at increasing
antibiotic concentrations. This effect can probably be attributed to addition of nitrates by
the compound itself rather than decreasing ability of the plants to assimilate nitrate into
its biomass due to antibiotic phytotoxicity.
Addition of sulfamethoxazole to the treatments without plants resulted in four
significant responses (Table 1). Increased nitrate concentrations were present for week
one and increased total nitrogen concentrations for weeks one through three. After one
24 week of exposure to sulfamethoxazole there was an increase in nitrate concentration in the growth medium. This would indicate that the antibiotic alone contributed nitrates.
This was also true with respect to total nitrogen concentration during all three weeks of exposure. There seems to be little or no effect, however, on plant treatments with respect to sulfamethoxazole. Week two was the only week when there was a significant response
(Figure 3). This response is similar to the one for total nitrogen during week two of the medium alone, indicating that any changes in total nitrogen concentrations can be attributed to the addition of the antibiotic. Therefore, it seems that sulfamethoxazole had little or no affect on the ability of Spirodela punctata to remove nitrogenous compounds.
Samples containing all three antibiotic compounds resulted in two significant responses when added to the mineral salt medium containing no plants (Table 1). During week two and three, the addition of the compounds resulted in increased nitrate concentrations at higher antibiotic concentrations when compared to the control.
Conversely, when exposed to samples containing S. punctata fronds, only week one resulted in significant nitrate values whereas week two and three did not (Figure 4).
Recalling the morphological effects that the combination of the antibiotics imposed on the plant (i.e. the “bleaching effect” which went away after two days of exposure), it would seem that fronds were able to adapt to the presence of the compounds or perhaps the combination of antibiotics resulted in subtractive effects.
Leaching of nutrients into the growth medium from decomposition of older fronds is not believed to be significant due to the sterile conditions in which the plants in this study were grown. Szabo et al. (2000) concluded that nutrient concentration increase
25 during decomposition of duckweed is primarily the result of microbial processes. Thus,
decomposition of fronds would occur at a much slower rate under aseptic conditions and
therefore presence of significant nitrogen concentrations in the medium is unlikely due to
the loss of these compounds by plant decay.
Overall, effects of these selected antibiotic compounds on ability of Spirodela
punctata to remove nitrogenous compounds from the growth medium were negligible.
Primarily, any differences in nitrogen concentrations were the result of addition of the antibiotics. The only exception was with total nitrogen concentrations in plant treatments containing chlortetracycline. There was a significant difference between all three weeks of exposure. There was no significant difference in samples containing medium and antibiotics alone.
Removal of nitrogenous compounds in wastewater by Spirodela punctata seems to be effective regardless of the presence of these antibiotic compounds. Growth inhibition resulting from inputs of the studied compounds at a level that impacts removal of nitrogen species would likely only occur at concentrations exceeding 300 µg/L.
However, antibiotic concentrations at these levels are not typical in wastewater. The concentrations of antibiotics in municipal wastewater are generally in the nanogram to microgram range with an average concentration approximately 50 µg/L.
26
LITERATURE CITED
American Society for Testing and Materials. 1998. Standard guide for conducting static toxicity tests with Lemna gibba. Annual Book of ASTM Standards, Vol. 11.05.
Bowker, D.W., A.N. Duffield, P. Denny. 1980. Methods for the isolation, sterilization, and cultivation of Lemnaceae. Freshwater Biology.
Boyd, C.E. 1969. Vascular aquatic plants for mineral nutrient removal from polluted waters. Economic Botany.
Brain, R.A., D.J. Johnson, S.M. Richards, H. Sanderson, P.K. Sibley, K.R. Solomon. 2003. Effects of 25 pharmaceutical compounds to Lemna gibba using a seven- day static-renewal test. Environmental Toxicology and Chemistry.
Brain R.A., D.J. Johnson, S.M. Richards, M.L. Hanson, H. Sanderson, M.W. Lam, C. Young, S.A. Mabury, P.K.Sibley, K.R. Solomon. 2004. Microcosm evaluation of the effects of eight pharmaceutical mixture to the aquatic macrophytes Lemna gibba and Myriophyllum sibiricum. Aquatic Toxicology.
Cheng, J., B.A. Bergmann, J.J. Classen, A.M. Stomp, J.W. Howard. 2001. Nutrient recovery from swine lagoon water by Spirodela punctata. Bioresource Technology.
Daughton, C.G., T.A. Ternes. 1999. Pharmaceuticals and personal care products in the environment: Agents of subtle change? Environmental Health Perspectives.
Ebringer, L. 1972. Are plastids derived from prokaryotic micro-organisms? Action of antibiotics on chloroplasts of Euglena gracilis. Journal of General microbiology.
Fleming, I., E. Vaclavik, B. Halling-Sorensen. 2003. Pharmaceuticals and personal care products: A source of endocrine disruption in the environment? Pure Applied Chemistry.
27
Halling-Sorensen, B., S. Nors Neilsen, P.F. Lanzky, F. Ingerslev, H.C. Holten Lutzhoff, S.E. Jorgensen. 1998. Occurrence, fate, and effects of pharmaceutical substances in the environment—A review. Chemosphere.
Harvey, R.M. and J.L. Fox, 1973. Nutrient removal using Lemna minor. Journal of Water Pollution Control Federation.
Hillman, W.T. and D.D. Cully. 1978. The uses of duckweed. American Scientist.
Huang, C.H., J.E. Renew, K.L. Smeby, K. Pinkston, and D.L. Sedlak. 2001. Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis. Water Resources Update.
Kummerer, K. 2001. Drugs in the environment: Emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources—A review. Chemosphere.
Jensen, J. 2001. Veterinary Medicines and Soil Quality: The Danish Situation as an Example. IN C. G. Daughton and T. L. Jones-Lepp (eds.) Pharmaceuticals and personal care products in the environment: scientific and regulatory issues. Washington D.C.: American Chemical Society.
Jones, O.A., N. Voulvoulis, J.S. Lester. 2001. Human pharmaceuticals in the aquatic environment a review. Environmental Technology.
Lillo, C., F. Provan, K.J. Appenroth. 1998. Photoreceptors involved in the regulation of nitrate reductase in Spirodela polyrhiza. Journal of Photochemistry and Photobiology B: Biology.
Mandi, L. 1994. Marrakesh wastewater purification experiment using vascular aquatic plants Eichhornia crassipes and Lemna gibba. Water Science Technology.
28 Margulis, L. 1981. Symbiosis in cell evolution. 1st Edition. Freeman, New York.
Marx, F. 2004. Small, basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression, structure, function and potential application. Applied Microbiological Biotechnology.
McFadden, G.I. and D.S. Roos. 1999. Apicomplexan plastids as drug targets. Trends in Microbiology.
Nickell, L. and P. Gordon. 1961. Effects of the tetracycline antibiotics, their degradation products, derivatives and some synthetic analogs on the growth of certain plant systems. Antimicrobial Agents Annual.
Pomati, F., A.G. Netting, D. Calamari, B.A. Neilan. 2004. Effects of erythromycin, tetracycline and ibuprofen on the growth of Synechocystis sp. and Lemna minor. Aquatic Toxicology.
Samuelsen, O.B., B.T. Lunestad, B. Husevag, T. Holleland, A. Ervik. 1992. Residues of oxolinic acid in wild fauna following medication in fish farms. Diseases of Aquatic Organisms Journal.
Sutton, D.L., W. H. Ornes. 1975. Growth of Spirodela polyrrhiza in static sewage effluent. Aquatic Botany.
Szabo, S., M. Braun, P. Nagy, S. Balazsy, O. Reisinger. 2000. Decomposition of duckweed (Lemna gibba) under axenic and microbial conditions: flux of nutrients between litter water and sediment, the impact of leaching and microbial degradation. Hydrobiologia.
Tripathi, B.D. and A.R. Upadhyay, 2003. Dairy effluent polishing by aquatic macrophytes. Water, Air, and Soil Pollution.
29 PERSONAL COMMUNICATION CITED
Mesler, M. 2006. Biology Professor. Humboldt State University.
30 APPENDIX A
Chlortetracyline
Lomefloxacin
Sulfamethoxazole