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Keywords:

  • Triclosan;
  • Biosolids;
  • Earthworms;
  • Nitrogen-cycling;
  • Respiration

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Triclosan (TCS) is a common constituent of personal care products and is frequently present in biosolids. Application of biosolids to land transfers significant amounts of TCS to soils. Because TCS is an antimicrobial and is toxic to some aquatic organisms, concern has arisen that TCS may adversely affect soil organisms. The objective of the present study was to investigate the toxicity and bioaccumulation potential of biosolids-borne TCS in terrestrial micro- and macro-organisms (earthworms). Studies were conducted in two biosolids-amended soils (sand, silty clay loam), following U.S. Environmental Protection Agency (U.S. EPA) guidelines. At the concentrations tested herein, microbial toxicity tests suggested no adverse effects of TCS on microbial respiration, ammonification, and nitrification. The no observed effect concentration for TCS for microbial processes was 10 mg/kg soil. Earthworm subchronic toxicity tests showed that biosolids-borne TCS was not toxic to earthworms at the concentrations tested herein. The estimated TCS earthworm lethal concentration (LC50) was greater than 1 mg/kg soil. Greater TCS accumulation was observed in earthworms incubated in a silty clay loam soil (bioaccumulation factor [BAF] = 12 ± 3.1) than in a sand (BAF = 6.5 ± 0.84). Field-collected earthworms had a significantly smaller BAF value (4.3 ± 0.7) than our laboratory values (6.5–12.0). The BAF values varied significantly with exposure conditions (e.g., soil characteristics, laboratory vs field conditions); however, a value of 10 represents a reasonable first approximation for risk assessment purposes. Environ. Toxicol. Chem. 2012;31:646–653. © 2011 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Triclosan (TCS; Irgasan DP 300 [trade name]; 5-chloro-2-[2, 4-dichloro phenoxy] phenol, CAS 3380-34-5) is a broad-spectrum antimicrobial agent and a common constituent of personal care products such as liquid soaps, detergents, toothpastes, and kitchenware. Triclosan is frequently detected in wastewater treatment residuals (biosolids) at parts-per-million concentrations 1, and biosolids land application can transfer biosolids-borne TCS to soils. A reported TCS degradation half-life is approximately 100 d 2; therefore, TCS can exist in biosolids-amended soils for extended times. Because TCS has antimicrobial properties and is toxic to some aquatic organisms 3, 4, concerns arise that TCS may adversely affect terrestrial organisms. Chemical toxicity depends on bioavailability that decreases with increasing contact time between the chemical and the soil 5. Decreased bioavailability makes the chemical's bioavailability assessment using total concentrations problematic. Thus, bioavailability assessments are generally performed using toxicity tests 6, which involve exposing test organisms to a chemical of interest at varying concentrations and subsequent monitoring of biological end-points (e.g., mortality, reproduction, growth, behavioral changes) over time.

Triclosan impacts on soil macro-organisms, especially earthworms (Eisenia foetida), are of particular interest because earthworms live in soil, and most of their diet consists of soil 7. Chemical exposure can occur through both dermal contact and soil ingestion. Furthermore, earthworms represent a significant portion of diet for many vertebrates such as birds, shrews (Blarina brevicauda), and herring gulls (Larus argentatus) 7, and they are often used as surrogates for soil-dwelling organisms in ecological risk assessments. Triclosan impacts on soil micro-organisms (e.g., bacteria) are also critical because they conduct vital ecological functions, notably facilitating organic matter decomposition and nitrogen cycling.

Significant knowledge gaps exist in our understanding of biosolids-borne TCS impacts on terrestrial organisms. Previous studies on TCS toxicity on earthworms 8, 9 and micro-organisms 10–12 were conducted in unamended (no biosolids) soils. Earthworm toxicity results were variable, with reported TCS toxic concentrations of greater than 1 mg/kg for genotoxic effects in silty clay loam soil 8, 1,026 mg/kg or less in artificial soil 9, and no adverse effects at 100 mg/kg or less in silty clay loam soil 8. Microbial processes (nitrification, respiration, enzyme activity) were inhibited at TCS concentrations of 5 to 50 mg/kg in two Australian soils (sand, organic carbon [OC] = 8.5 g/kg; clay, OC = 18.5 g/kg). Butler et al. 11 also suggested microbial respiration (basal and substrate induced) inhibition at 10 mg/kg TCS in three soils (sandy loam, OC = 17 g/kg; loamy sand, OC = 27 g/kg; clay, OC = 23 g/kg), but noted that the inhibition disappeared within 7 d of initial spiking. In contrast, Reiss et al. 12 reported no inhibitory effects of TCS (≤2 mg/kg sandy loam soil) on nitrification and microbial respiration. These studies 8–12 involved spiking of TCS to soils rather than biosolids. Potential toxicity impacts of biosolids-borne TCS to terrestrial organisms are unknown. Triclosan may be less toxic when present as an inherent component of biosolids, because TCS is hydrophobic (log Koc = 4.2, 13) and tends to strongly partition to the solid fraction of biosolids, which reduces its bioavailability.

Besides being toxic to some aquatic organisms, TCS bioaccumulates in algae and snails 14, 15 (bioaccumulation factor [BAF] ≥ 1,000) and in earthworms collected from biosolids-amended soils 16, 17. Bioaccumulation in soil organisms may have implications for biomagnification in organisms higher in the food chain. Kinney et al. 16 suggested significant bioaccumulation (BAF = 10.8 and 27) in earthworms collected 31 and 156 d after biosolids application, but the results were confounded because of the detection of significant TCS concentration (0.88 mg/kg) even in the control (no reported amendments for last 7 years). Higgins et al. 17 measured TCS bioaccumulation in earthworms exposed to biosolids-borne TCS in field soils (fine sand, silty clay loam). Estimated BAFs were based on only a few samples, and internal inconsistencies in the data led to inconclusive results. Limited data exist on the bioaccumulation and toxicity of biosolids-borne TCS to terrestrial organisms. These data are critical to assess the ecological health risk of biosolids-borne TCS on soil-dwelling organisms.

We hypothesize that biosolids-borne TCS may accumulate in earthworms but is not toxic to micro- and macro-organisms. The objectives of the present study were to investigate the toxicity of biosolids-borne TCS to terrestrial macro- (earthworms) and micro-organisms and also to explore the bioaccumulation potential in earthworms. The laboratory toxicity tests were conducted following the Office of Prevention, Pesticides, and Toxic Substances Soil Microbial Community Toxicity Test (Guideline 850.5100) 18 and Earthworm Sub-chronic Toxicity Test (Guideline 850.6200) 19. The guidelines required range-finding and definitive tests and prescribed adding the chemical of interest directly to a natural soil. The protocol was modified herein to deliver TCS as a component of biosolids in an effort to better mimic the primary mechanism of TCS transfer to the soil through biosolids. The microbial toxicity test quantified the effects of TCS on the processes of microbial respiration, ammonification, and nitrification. The earthworm test quantified the TCS toxicity, using mortality as an indicator. In addition, an earthworm bioaccumulation test was conducted concurrently with the earthworm toxicity test. To assess the longer-term bioavailability of TCS and to compare with our laboratory measurements, we also measured bioaccumulation in earthworms collected from a biosolids-amended field site and a representative control site.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Chemicals, biosolids, and soils

Triclosan (CAS No. 101-20-2; >99.9% purity) standard was purchased from United States Pharmacopeia. Triclosan internal standard (13C12-TCS), pyridine, bis (trimethylsilyl) trifluoroacetamide + 1% trimethylchloro silane, potassium chloride (KCl), potassium hydroxide (KOH), sodium hydroxide (NaOH), barium chloride (BaCl2), hydrochloric acid (HCl), nitrate (NOmath image) standard, ammonium (NHmath image) standard, and solvents (methanol [MeOH], acetone, hexanes, dichloromethane) of high-performance liquid chromatography grade or greater were purchased from Sigma Aldrich, JT Baker, or Fisher Scientific. Anaerobically digested biosolids (solids = 320 g/kg) were collected from a domestic wastewater treatment plant in Illinois. The biosolids contained 5 mg/kg TCS. Two soils, an Immokalee fine sand (IFS) (sandy, siliceous, hyperthermic Arenic Alaquods) and the Ashkum silty clay loam (ASL) (fine, mixed, superactive, mesic Typic Endoaquolls) were collected in 2008 from sites with no known history of receiving land-applied biosolids or wastewater treatment sludge. An artificial soil (68% silica sand, 20% kaolin clay, 10% sphagnum peat moss, 2% calcium carbonate; all by weight) was prepared by mixing the various ingredients as prescribed by U.S. Environmental Protection Agency (U.S. EPA) guidelines 20 and used in the earthworm toxicity test along with two natural soils. The major physicochemical properties of the soils and biosolids used are described in Table 1. The earthworms were purchased from Carolina Biological. Before use, the earthworms were grown in moist peat moss (growing medium) and fed with worm food (Magic products) each day for 21 d.

Table 1. Major physico-chemical properties of the soils and biosolids used in the present study
SoilTextureOrganic carbon (g/kg)pH (1:1)
  1. IFS = Immokalee fine sand; ASL = Ashkum silty clay loam.

IFSSand114.5
ASLSilty clay loam346.6
Artificial soilPeat moss,10% Ca carbonate, 2%487.7
Biosolids2508.0
Field control soilClay loam407.8
Field amended soilSilty clay loam807.1

Microbial toxicity test design

The microbial toxicity test included a range-finding and definitive test. The unreplicated range-finding test was conducted in both IFS and ASL soils and identified the appropriate range of TCS concentrations for a subsequent definitive test. The TCS concentrations used in the range-finding test were 5 to 10,005 mg/kg biosolids (soil concentrations = 0.05 to 100 mg/kg; biosolids application rate = 22 Mg/ha). Triclosan was added to the biosolids using MeOH as a carrier solvent. A no-carrier solvent biosolids-amended soil control was included to quantify solvent addition effects. The range-finding test identified possible adverse effects on NO3-N + NO2-N production at TCS concentrations ≥ 0.15 mg TCS/kg soil and on carbon dioxide (CO2) evolution at 10 mg/kg soil. Solvent addition did not adversely affect any reaction. Based on the range-finding test, a TCS concentration range of 0.05 to 10 mg/kg soil was selected for a definitive test.

The definitive test included 1-g samples of dried biosolids spiked with various TCS concentrations using MeOH as a carrier solvent. The spiked biosolids were subsequently air-dried to allow carrier solvent evaporation, rewetted with water, and equilibrated for 48 h in 300-ml glass Mason jars. Three replicates were prepared for each spiked concentration, soil-only control (no biosolids or TCS spike), and for each sampling time. Spiked biosolids were amended to 100 g (dry wt) of IFS and ASL soils (equivalent biosolids application rate of 22 Mg/ha), and amended soils were brought to field capacity (10% for IFS and 30% for ASL by weight). Treatment spikes were in addition to the inherent biosolids TCS concentration, resulting in nominal final TCS concentrations of 0.05, 0.15, 1, 5, and 10 mg/kg soil.

The sample jars were aerated with CO2-free air at 22°C. Incoming air was stripped of CO2 and humidified by pumping ambient air first through 2 M KOH, followed by CO2-free water, a column of soda lime chips (Ca [OH]2 > 80%, KOH < 3%, NaOH < 2%, ethyl violet < 1%), additional 2 M KOH, and once more through CO2-free water. The CO2 evolved from each sample jar (measure of microbial respiration) was collected in a series of two base traps, each containing 100 ml 0.15 M KOH. Base traps connected to the samples were removed and replaced with fresh base at 5 d and 28 d, and analyzed for CO220. Subsets (10 g dry wt) of amended soil were sampled at 0, 5, 14, and 28 d and shaken with 100 ml 1 M KCl 21. Extracts were filtered (0.45 µm) and analyzed for NO3-N + NO2-N 22 and NH4-N 23 to assess TCS impacts on nitrification and ammonification, respectively. Results are reported as milligrams NO3-N + NO2-N and NH4-N per kg soil or biosolids-amended soil. A rapid flow analyzer (Alpkem) was used for NO3-N + NO2-N and AQ2 discrete analyzer for NH4-N analyses.

Earthworm toxicity test design

Similar to the microbial toxicity test, the range-finding earthworm toxicity test design included a TCS concentration range of 5 to 10,005 mg/kg biosolids, but three soils (IFS, ASL, and artificial) rather than two. Earthworms in the IFS soil were adversely affected by the TCS addition. At 28 d, nearly 100% mortality occurred at TCS concentrations >5 mg/kg biosolids, whereas concentrations of 10,005 mg/kg or less had minimal adverse effect on earthworm survival in the ASL and artificial soils. Based on the range-finding test results, the definitive test only included the IFS soil and a TCS concentration range of 5 to 105 mg/kg biosolids (soil concentration = 0.05–1 mg/kg). The definitive test included soil-only controls (no biosolids or TCS spike) and biosolids samples (in quadruplicate) spiked with various TCS concentrations. Spiked biosolids were dried to remove solvent, rewetted with water, equilibrated for 48 h, and amended to 200 g (dry wt) of the IFS soil (equivalent biosolids application rate of 22 Mg/ha) in 800-ml glass Mason jars. The test also included no solvent biosolids-amended controls (no additional TCS spike) to quantify carrier solvent effect. The final nominal TCS concentrations were 0.05, 0.07, 0.10, 0.15, 0.55, and 1 mg/kg soil.

Earthworms were removed from the growing medium and washed with deionized distilled water before using them in the toxicity test. Soil was brought to field capacity (10% by weight), and 10 earthworms were added to each sample. Lids were placed loosely on top of the incubation jars to reduce moisture loss and to prevent earthworm escape. Dead earthworms at the soil surface were counted and removed as necessary each day, and the numbers of living earthworms were tallied every 7 d up to 28 d.

Earthworm bioaccumulation laboratory study

An earthworm TCS bioaccumulation study was run concurrently with the definitive earthworm toxicity test. A lower TCS concentration range was selected, because live earthworms were required for quantifying TCS accumulation. Two-gram samples of biosolids were spiked with TCS concentrations of 0, 2.5, 5, 10, 50, and 100 mg/kg of biosolids and amended to the IFS and ASL soils, in quadruplicate as in the definitive toxicity test. The nominal final soil TCS concentrations were 0.05, 0.07, 0.10, 0.15, 0.55, and 1 mg/kg of soils. After 28 d, earthworms were removed, counted, washed, and weighed. The earthworms were allowed to depurate for 24 h in petri dishes lined with moistened filter paper 24 and were subsequently frozen until analyses. Birds and worm-eating animals feed on nondepurated earthworms, but depuration allows earthworms to excrete TCS-contaminated soil or organic matter remaining in the gut. Thus, the measured TCS concentration in earthworms reflects accumulation in the tissue, rather than TCS sorbed to the soil present in the gut.

Earthworm bioaccumulation field study

Longer-term bioavailability of biosolids-borne TCS was estimated by using earthworms and corresponding soil samples collected from a field site in Illinois. The site received a single application of biosolids (application rate = 228 Mg/ha, 10-fold the agronomic rate) in 2008. Biosolids were incorporated to a soil depth of 15 to 20 cm, and the amended soil was used to grow grass (Poa pratensis) through the sample collection in 2010. Although the biosolids application rates were much greater than the agronomic rates, no evidence was seen of adverse effects on the growth of grass. In 2010, the earthworms were collected from the biosolids-amended site and the control site (18 m apart; similar soil texture) that had no history of biosolids application. The earthworms were collected using the hot mustard extraction method 25. Briefly, a mustard (allyl isothiocyanate) solution was applied to the soil surface that encouraged earthworm emergence to escape the mustard's irritant properties. The method was efficient in collecting a consistent number (≥20 from each location) of earthworms and did not require digging or hand sorting. The collected worms were shipped to our Florida laboratory in aerated bags with wet peat moss under ice packs. On arrival, earthworms were cleaned with deionized distilled water to remove peat moss residue, kept in petri dishes with wet filter paper, and allowed to depurate for 24 h.

Sample extraction and derivatization for bioaccumulation study

A sample extraction technique was modeled after previously described methods 26, 27 with few modifications. Frozen earthworms from the laboratory experiments were thawed, and fresh earthworms from the field were transferred to aluminum weigh boats, dried at 50°C to a constant weight, and ground. The dried and ground tissue (0.5–1 g) was added into 25-ml glass tubes. Solvent controls (containing no TCS) and untreated earthworms (spiked with known TCS concentrations) were subjected to the same extraction procedure to confirm that TCS was not introduced during the extraction procedure and to determine extraction method recoveries of TCS. The earthworm tissues were spiked with 13C12-TCS internal standard and extracted (twice) using a solvent mixture (10 ml) of MeOH + acetone (50:50, v/v), shaken on a platform shaker for 18 h, followed by 60 min sonication (Branson 2210; 40°C, 60 sonications/min) in a water bath. Suspensions were centrifuged at 800 g and the supernatant transferred to 20-ml glass scintillation vials. The two extracts were combined and dried under a gentle N2 stream. The dried extracts were reconstituted in 1 ml MeOH and transferred to 2-ml microcentrifuge tubes and centrifuged at 18,000 g for 30 min. The supernatant was transferred to 1-ml gas chromatograph vials and dried under a mild N2 stream. Triclosan derivatization was performed according to the method by Shareef et al. 28 with slight modification. Briefly, the dried supernatant was reconstituted in a mixture of 4:1 of derivatization agent (bis [trimethylsilyl] trifluoroacetamide + 1% trimethylchloro silane) and the solvent (pyridine), vortexed for 10 s, and heated in a dry bath for 1 h. The samples were then transferred to fresh gas chromatograph vials with glass inserts and Teflon-lined caps. The average percentage of recovery for the TCS spiked into earthworms was 90 ± 4.5%. The samples obtained after the derivatization step were analyzed by splitless injection (5 µl) on a Varian 4000 gas chromatograph equipped with Restek Rxi-5Sil column coupled with Varian 4000 MS/MS. Details of the instrumental conditions and limits of detection are presented in the Supplemental Data.

Statistical analysis

Statistical analysis was performed using SAS software, Version 9.1 29. The Tukey's studentized range test was used to assess statistical differences at each sampling period across treatments. Regression analysis with the SAS ESTIMATE procedure assessed the statistical differences between sampling times within treatments.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Impacts on soil microbial respiration

Cumulative CO2 evolution increased with time in all the treatments involving the IFS soil. Over 28 d, more CO2 was produced in biosolids-amended soils (230–320 mg) than the control (130 mg), irrespective of the amended soil TCS concentration (Fig. 1). The trend was attributed to increased substrate-induced microbial respiration following the addition of labile OC via the biosolids. Barbarick et al. 30 observed increased rates of microbial respiration in shrubland and grassland soils amended with biosolids (30–40 Mg/ha) for six years and attributed the increase to the availability of additional carbon substrates furnished by biosolids addition. Sullivan et al. 31 observed enhanced CO2 production and metabolism of the microbial community in a semi-arid rangeland soil (sandy loam, pH = 6.2) even 12 years after biosolids application.

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Figure 1. Mean total carbon dioxide (CO2) (mg) ± standard deviation evolved as a function of triclosan (TCS) concentrations in amended soils and time (0–28 d) in the Immokalee fine sand (IFS) soil treatments (like letters indicate no significant difference among treatments). The soil-only control represents the soil with no amendment of biosolids or TCS.

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At 28 d, the greatest cumulative CO2 evolution occurred in the treatment representing a TCS concentration of 10 mg/kg soil, but it was not significantly different from CO2 evolution from other TCS treatments (Fig. 1). At all times, the smallest amount of CO2 was evolved at the TCS concentration of 0.15 mg/kg treatment and likely represented an unexplained experimental error. Biosolids-amended soil TCS concentrations of 10 mg/kg soil or less did not adversely affect the CO2 evolution in the IFS soil (Fig. 1) or in the ASL soil (data not shown). Waller and Kookana 10 found no negative effect of TCS on substrate-induced respiration at concentrations less than 50 mg/kg soil. Butler et al. 11 spiked TCS (0–1,000 mg/kg) in sandy loam (OC = 17 g/kg), clay (OC = 7 g/kg), and loamy sand (OC = 23 g/kg) soils. Basal and substrate-induced respiration were inhibited at TCS concentrations greater than 10 mg/kg soil within 6 h of spiking, but the inhibition disappeared after 7 d. Butler et al. 11 speculated that TCS may have inhibited initial respiration (6 h) but that respiked TCS acted as a carbon source and stimulated respiration. Thus, the stimulation of microbial respiration observed in the present study could be a combined effect of TCS and biosolids serving as carbon sources.

Impacts on ammonification and nitrification (N-cycling)

In the IFS soil, greater NH4-N was released after biosolids or TCS addition than in the soil-only control (Fig. 2A). At 5 d, soil with a TCS concentration of 10 mg/kg had the greatest NH4-N concentration but was not significantly different from other TCS treatments. At study termination (28 d), biosolids addition significantly increased the NH4-N release, but no TCS concentration effect occurred, except a reduction in NH4-N concentration at 5 d in the 5-mg/kg treatment (Fig. 2A). Consistently, Holt et al. 32 observed increased NH4-N concentration up to 42 d after biosolids addition (18 Mg/ha) to a soil (unknown texture). Greater NH4-N concentration could be attributed to the addition of NH4-N from biosolids (at time zero) 33 and ammonification of added biosolids-organic N with time 32.

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Figure 2. Mean NH4-N (A) and NO3-N + NO2-N (B) concentrations (n = 3) ± standard deviation as a function of triclosan (TCS) concentrations in amended soil and time (0–28 d) in the IFS soil treatments (like letters indicate no significant difference among treatments). The soil-only control represents the soil with no amendment of biosolids or TCS.

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At 5 d, NO3-N + NO2-N release was similar in control and biosolids-amended treatments, suggesting no effect of biosolids or TCS addition. At 14 d, some inhibition occurred at a TCS concentration of 1 and 10 mg/kg; however, the inhibition disappeared at a concentration of 1 mg/kg at day 28. At 28 d, NO3-N + NO2-N concentrations were significantly smaller in the biosolids treatments than the soil-only control, but concentrations were independent of TCS additions except at a TCS concentration of 10 mg/kg (Fig. 2B). Decreased NO3-N + NO2-N production may reflect denitrification, possibly associated with anoxic microsites in biosolids-amended soil 27. Previous assessments (http://www.nwbiosolids.org/pubs/ManagingNitrogen.pdf) suggest that biosolids can promote denitrification by supplying ample quantities of labile OC and NO3-N, and by increasing the probability of anaerobic microsites formation. Thus, the balance between the NH4-N and NO3-N + NO2-N production (N-cycling) appears to be a function of biosolids addition, but it is unaffected by TCS concentration.

In summary, the CO2 evolution, NH4-N, and NO3-N + NO2-N release data suggest significant biosolids addition effects, but no TCS addition effects at concentrations ≤10 mg/kg soil in the IFS soil or the ASL soil (data not shown). The no observed effect concentration for TCS for microbial reactions would be at least 10 mg/kg soil (equivalent to 1,000 mg/kg biosolids). The TCS toxicity level in the amended soil would require an application of biosolids with typical TCS concentrations (10–20 mg/kg biosolids) for 50 to 100 years (assuming no TCS loss-degradation).

Earthworm toxicity test (IFS soil)

Biosolids-borne TCS additions did not affect earthworm survival. The mean earthworm survival was more than 90% in all TCS treatments, as well as in the control, except at 0.10 and 0.15 mg/kg soils (Fig. 3). The soil-only control appeared to provide sufficient nutrients for earthworm survival up to 28 d. No adverse TCS treatments, biosolids additions, or carrier solvent addition effects on the survival of earthworms were seen. Survival was affected at TCS concentrations 0.10 to 1 mg/kg soils, but no statistical difference was seen among the treatments because of the large variability (Fig. 3). Thus, no adverse effect was observed on the earthworm survival at TCS soil concentrations of 1 mg/kg or less. A definitive earthworm lethal concentration (LC50) value cannot be calculated from the data, because no significant adverse effect occurred up to the maximum tested concentration. An estimated LC50 value in the IFS soil was greater than 1 mg TCS/kg (equivalent to a biosolids concentration of >100 mg/kg). Data from the range-finding test can be used to estimate an LC50 of greater than 100 mg/kg soils (equivalent biosolids TCS concentration >10,000 mg/kg) in the ASL and artificial soils. The toxic levels estimated herein are much greater than the typical TCS concentrations (mean = 16 mg/kg, 95th percentile = 62 mg/kg) in biosolids 1.

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Figure 3. The mean earthworm survival (%) (n = 4) ± standard deviation as affected by amended soil triclosan (TCS) concentrations and the duration of earthworm exposure in the Immokalee fine sand (IFS). The soil-only control represents the soil with no amendment of biosolids or TCS.

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Triclosan toxicity data (LC50 > 1 mg/kg soils) are consistent with previously reported no adverse effects at TCS concentration of 100 mg/kg soil or less; however, genotoxic effects were reported at 1 mg/kg in one study 8. The TCS toxicity level in the amended soil would require an application of biosolids with typical TCS concentrations (10–20 mg/kg biosolids) for 5 to 10 years (assuming no TCS loss-degradation).

Earthworm bioaccumulation laboratory study

Triclosan bioaccumulation was assessed through the calculation of BAF values, expressed as the ratio of TCS concentration in the earthworm tissue to the TCS concentration in the exposure soil. Bioaccumulation data suggested that biosolids-borne TCS accumulated in the earthworm tissues. The average BAFs in the two soils, irrespective of the spiked TCS concentration, were 6.5 ± 0.84 for the IFS soil and 12 ± 3.08 for the ASL soil (Table 2). The average measured BAFs in the two soils were significantly different (p < 0.05). The difference was attributed to differences in soil OC contents (11 g/kg for IFS soil and 34 g/kg for ASL soil; Table 1), with a greater TCS accumulation by earthworms in high OC soil (ASL). In the present study, application of biosolids (at 22 Mg/ha) having an OC content of 250 g/kg added an additional 2.7 g/kg of OC to both soils, but the total OC was still smaller in the amended IFS soil (13.7 g/kg) than in the amended ASL soil (36.7 g/kg). The bioaccumulation trend may reflect earthworms' preferential feeding on OC and ingesting both OC and TCS that were associated with the biosolids matrix (i.e., with OC). Our results are consistent with a previous bioaccumulation study 27 conducted for a similar antimicrobial compound triclocarban. The measured BAF values of triclocarban were greater, but not significantly so, in a similar loam soil (20 ± 2.1) than in the IFS (18 ± 3.5) soil. The BAFs obtained in the present study were generally not a function of TCS concentration. For example, in the IFS soil, the BAFs did not vary significantly with TCS treatments (Table 2). However, in the ASL soil, BAFs increased as TCS concentrations increased with significantly greater accumulation at 0.15 mg/kg soil, but the accumulation did not differ among the other TCS treatments (Table 2).

Table 2. Measured and model (BASL4) estimated TCS concentrations (means; n = 3 or 4 and standard error), BAF, and BSAF in the earthworm tissue in IFS and ASL soils
MeasuredBASL4 estimated
IFS soilASL soilIFS soilASL soil
Nominal TCS soil concentration in amended soils (mg/kg)Earthworm tissue concentration (mg/kg dry wt)BAFa (gsoil/gtissue)BSAF (goc/glipid)Nominal TCS soil concentration in amended soils (mg/kg)Earthworm tissue concentration (mg/kg dry wt)BAFa (gsoil/gtissue)BSAF (goc/glipid)BAF (gsoil/gtissue)BAF (gsoil/gtissue)
  • a

    Like letters represent no statistical difference at 5% significance level among treatments.

  • b

    Limit of quantitation (LOQ) is measured at a signal/noise ratio of < 10. The LOQ of TCS was 0.7 mg/kg for the earthworm tissue. The concentrations <LOQ were estimated as ½ the LOQ of the instrument.

  • c

    Statistical difference at 5% level of significance.

    BASL4 = Biosolids-Amended Soil Level IV; TCS = triclosan; BAF = bioaccumulation factor; BSAF = biota-sediment accumulation factor; IFS = Immokalee fine sand; ASL = Ashkum silty clay loam.

0.05<LOQb<72.40.05<LOQb<14 b7.45.02.2
0.0750.6 ± 0.07.3 ± 1.9 a2.30.075<LOQb<4.7 b4.95.02.2
0.100.5 ± 0.14.9 ± 1.4 a1.70.101.1 ± 0.17.4 ± 2.3 b11.75.02.2
0.150.6 ± 0.14.3 ± 1.4 a1.40.151.6 ± 0.318 ± 1.3 c11.35.02.2
0.554.9 ± 0.58.8 ± 0.9 a3.1112.6 ± 0.712 ± 0.7 b12.75.02.2
17.3 ± 1.07.3 ± 1.0 a2.45.0
Average 6.5 ± 0.84c2.2 ± 0.59  12 ± 3.1c9.6 ± 3.35.0 ± 0.02.2 ± 0.0

Earthworm bioaccumulation field study

Field-collected worms also accumulated the biosolids-borne TCS. The TCS concentrations in earthworms collected from three locations within the amended field soil varied from 2.6 to 5.9 mg/kg, perhaps reflecting varying earthworm activity patterns in the soil. Earthworms occupy a range of soil depths, depending on the season, species, and life history stage, and they may be exposed to varying amounts of TCS. Grab soil samples collected from the same area where the earthworms were collected had average TCS concentrations of 0.99 mg/kg amended soil. The average BAF value from the three locations was 4.3 ± 0.7 (Table 3). Triclosan concentrations were nondetectable (<0.22 mg/kg) in earthworms and soil collected from a representative control site.

Table 3. Measured TCS concentrations (means [mg/kg]; n = 3 or 4 and standard error), BAF, and BSAF ± SE in the earthworms collected from the field equilibrated biosolids-amended soils (like letters represent no statistical difference among treatments)
 Inherent amended soil concentrationMeasured earthworm tissue concentrationBAF (gsoil/gtissue)BSAF (goc/glipid)
mg/kg
  1. TCS = triclosan; BAF = bioaccumulation factor; BSAF = biota-sediment accumulation factor.

Location 10.992.6 ± 0.22.7 ± 0.2 a6.6
Location 20.994.4 ± 2.54.4 ± 2.5 a11
Location 30.995.9 ± 0.95.9 ± 0.9 a15
 Overall average4.3 ± 1.94.3 ± 0.710.9 ± 4.2

The texture of the field soil was similar to the ASL soil used in our laboratory study, but the BAF values for the field earthworms (4.3 ± 1.9) were significantly smaller (p < 0.05) than the average values for the ASL soil in the laboratory study (12 ± 3.1). The difference may be attributed to a variety of reasons. Triclosan availability may be greater under laboratory conditions because of lower OC content of the laboratory soil (OC = 34 g/kg) than the field soil (OC = 80 g/kg), and also that TCS was spiked to the amended laboratory soils as opposed to being an inherent component of biosolids applied in the field soil. Field-collected worms are believed to belong to multiple species; chemical bioaccumulation may vary with the earthworm species, which could be related to differences in feeding habits, chemical elimination rates, and species weights. Furthermore, field temperature and moisture conditions vary more appreciably than under controlled laboratory conditions. Field earthworms are exposed to much larger soil volumes and burrow to deeper or shallower depths, depending on the species 34, which may reduce exposure and TCS accumulation potential.

Comparison of measured and modeled bioaccumulation potential

The partitioning theory for traditional hydrophobic organic contaminants is commonly used to predict the partitioning of chemicals to invertebrates from sediments 26. The biota-sediment accumulation factor (BSAF) is calculated from the lipid normalized earthworm and OC normalized soil chemical concentrations (Eqn. 1).

  • equation image(1)

The Corg is the lipid-normalized earthworm chemical concentration; Csoil is the OC-normalized soil chemical concentration. The flip (fraction of lipid) in the earthworm was 0.032 ± 0.01 17, and fOC (fraction of OC) was 0.011 for IFS soil, 0.034 for ASL soil, and 0.08 for the field biosolids-amended soil (Table 1). According to Equation 1, the estimated earthworm BSAF values are 2.2 ± 0.59 in the IFS soil, 9.6 ± 3.3 in the ASL soil (Table 2), and 10.9 ± 4.2 in the field-collected soils (Table 3). The hydrophobic organic contaminants theory predicts a BSAF of approximately 1.6 for nonmetabolized organic compounds if the log Kow of a compound is less than six 35. The estimated log Kow of TCS is 4.8, but the TCS BAF values were very different. The difference between expected and estimated BSAF values suggests that hydrophobic organic contaminants theory does not accurately predict the TCS accumulation in earthworms. The theory assumes equilibrium between the soil solid and water phases, which is often not true. Furthermore, the estimated BSAF value is similar to the expected value when the earthworms acquire the chemical only from the soil solution. In soils, however, the earthworms can accumulate TCS from the soil solution, direct ingestion of soil or biosolids and from direct partitioning of biosolids-borne TCS to the earthworm tissue 16.

We also used the Biosolids-Amended Soil Level IV (BASL4) model 36 to estimate earthworm bioaccumulation. This model uses the concept of fugacity and models chemical distribution in soil by assuming that the growing medium could be either at equilibrium, steady-state, or nonsteady state. The model parameters were selected based on the physic-chemical properties of TCS and soil type where the organisms were grown. The BASL4 assumes that the properties of invertebrates and mammals are representative of earthworms and shrews, respectively 37. The BASL4-predicted average BAF value for the IFS soil (5.0 ± 0.0) was reasonably close to our laboratory measured value of 6.5 ± 0.84 (Table 2). The predicted value (2.2 ± 0.0) for the ASL soil was much less than the laboratory-measured value of 12 ± 3.1 (Table 2).

The modeled values (BASL4) generally underestimated the bioaccumulation potential of TCS by earthworms, because the models assume bioaccumulation to occur only from soil pore water. Earthworm bioaccumulation of TCS also does not appear to follow the traditional hydrophobic organic contaminants theory. Thus, the various estimates should be used with caution. The measured BAFs (4.3–12) obtained herein were smaller than previously determined values (10.8–27) by Kinney et al. 16, and thus, the potential for biomagnification in higher organisms (e.g., predators) is expected to be less than that hypothesized by Kinney et al. 16. We conclude that the measured and modeled BAFs are in the same order of magnitude, and that a reasonable first approximation of the TCS earthworm BAF value is 10 across soils, environmental conditions, and perhaps earthworm species.

SUMMARY AND CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Triclosan treatment (≤10 mg/kg soil) did not adversely affect microbial respiration, nitrification, or ammonification (N-cycling), and the suggested no observed effect concentration is 10 mg/kg soil. Biosolids-borne TCS is not toxic to earthworms. The estimated LC50 for earthworms was at least the highest TCS concentration tested herein (>1 mg/kg soil), because no adverse impacts were observed at the highest TCS concentration. Biosolids containing typical TCS concentrations (10–20 mg/kg) applied at agronomic rates (∼22 Mg/ha) yield amended soil TCS concentrations of 0.10 to 0.20 mg/kg. Thus, typical biosolids can be land applied (considering no TCS loss) for 5 to 10 years without causing earthworm toxicity and for 50 to 100 years without causing adverse effects on microbial reactions (respiration, nitrification, or ammonification). Assuming a half-life of 100 d, amended soil TCS concentrations never reach levels potentially harmful to terrestrial organisms (earthworms, micro-organisms). Triclosan bioaccumulation in earthworm tissue varied between laboratory and field conditions, with physicochemical properties of soils and whether bioaccumulation was measured or estimated (using models). Most of the measured BAF values, however, are of the same order of magnitude, and a BAF value of 10 represents a reasonable first approximation of bioaccumulation potential. Significance of an earthworm BAF value of 10 can be best assessed in a risk assessment addressing the earthworm-eating predator pathway and will be the subject of a subsequent paper.

The earthworm toxicity values determined herein can be regarded as estimates, because of the lack of adverse effects at the highest concentration tested. Future investigations could include long-term TCS impact studies or studies using greater TCS concentrations, although greater concentrations are environmentally unrealistic. The microbial community structure changes and earthworm sublethal effects on parameters such as growth inhibition, reproduction (cocoon and juvenile counts), burrowing behavior, and protein content were not investigated herein and could also be topics of future research.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

This research was made possible through a grant from Metropolitan Water Reclamation District of Greater Chicago (requisition # 1211601). We thank L. Hundal and K. Kumar for help in collecting and shipping the field earthworms. We also thank C. Wilson for providing access to and help in performing the gas chromatography analytical work.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information
  • 1
    U.S. Environmental Protection Agency. 2009. Targeted National Sewage Sludge Survey Sampling and Analysis Technical Report. EPA-822-R-08-016. Technical Report. Washington, DC.
  • 2
    Waria M, O'Connor GA, Toor GS. 2011. Biodegradation of Triclosan (TCS) in biosolids-amended soils. Environ Toxicol Chem 30: 24882496.
  • 3
    Veldhoen N, Skirrow RC, Osachoff H, Wigmore H, Clapson DJ, Gunderson MP, Aggelen GV, Helbing CC. 2006. The bactericidal agent triclosan modulates thyroid hormone-associated gene expression and disrupts postembryonic anuran development. Aquatic Toxicol 80: 217227.
  • 4
    Yang LH, Ying GG, Su HC, Stauber JL, Adams MS, Binet MT. 2008. Growth-inhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga Pseudokirchneriella subcapitata. Environ Toxicol Chem 27: 12011208.
  • 5
    Alexander M. 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ Sci Technol 34: 42594265.
  • 6
    Singh A, Ward OP. 2004. Biodegradation and Bioremediation. Springer, Berlin, Germany.
  • 7
    Suter GW, Efroymson RA, Sample BE, Jones DS. 2000. Ecological Risk Assessment for Contaminated Sites. CRC, Boca Raton, Florida, USA.
  • 8
    Lin D, Zhou Q, Xie X, Liu Y. 2010. Potential biochemical and genetic toxicity of triclosan as an emerging pollutant on earthworms (Eisenia fetida). Chemosphere 81: 13281333.
  • 9
    Samsoe-Petersen L, Winther-Nielsen M, Madsen T. 2003. Fate and effects of triclosan. Project 861. Danish Environmental Protection Agency, Copenhagen, Denmark.
  • 10
    Waller NJ, Kookana RS. 2009. Effect of triclosan on microbial activity in Australian soils. Environ Toxicol Chem 28: 6570.
  • 11
    Butler E, Whelan MJ, Ritz K, Sakrabani R, van Egmond R. 2011. Effects of triclosan on soil microbial respiration. Environ Toxicol Chem 30: 360366.
  • 12
    Reiss R, Lewis G, Griffin J. 2009. An ecological risk assessment for triclosan in the terrestrial environment. Environ Toxicol Chem 28: 15461556.
  • 13
    Agyin-Birikorang S, Miller M, O'Connor GA. 2010. Retention and release of TCC and TCS in soils and sediments. Environ Toxicol Chem 29: 19251933.
  • 14
    Coogan MA, Edziyie RE, La Point TW, Venables BJ. 2007. Algal bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater treatment plant receiving stream. Chemosphere 10: 19111918.
  • 15
    Coogan MA, La Point TW. 2008. Snail bioaccumulation of triclocarban, triclosan, and methyltriclosan in a North Texas, USA, stream affected by wastewater treatment plant runoff. Environ Toxicol Chem 27: 17881793.
  • 16
    Kinney CA, Furlong ET, Kolpin DW, Burkhardt MR, Zaugg SD, Werner SL, Bossio JP, Benotti MJ. 2008. Bioaccumulation of pharmaceuticals and other anthropogenic waste indicators in earthworms from agricultural soil amended with biosolids or swine manure. Environ Sci Technol 42: 18631870.
  • 17
    Higgins CP, Paesani ZJ, Abbott Chalew TE, Halden RU, Hundal LS. 2011. Persistence of triclocarban and triclosan in soils after land application of biosolids and bioaccumulation in Eisenia foetida. Environ Toxicol Chem 30: 556563.
  • 18
    U.S. Environmental Protection Agency. 1996. Product Properties Test Guidelines: OPPTS 850.5100, Soil Microbial Community Toxicity Test. Washington, DC.
  • 19
    U.S. Environmental Protection Agency. 1996. Product Properties Test Guidelines: OPPTS 850.6200, Earthworm Subchronic Toxicity Test. Washington, DC.
  • 20
    Anderson JP. 1982. Soil respiration. In Page AL, Miller RH, Keeney DR, eds, Methods of Soil Analysis, Part 2—Chemical and Microbiological Properties, 2nd ed. ASA and SSSA, Madison, WI, USA, pp 842843.
  • 21
    Keeney DR, Nelson DW. 1982. Nitrogen-Inorganic forms. In Page AL, Miller RH, Keeney DR, eds, Methods of Soil Analysis. Part 2—Chemical and Microbiological Properties, 2nd ed. ASA and SSSA, Madison, WI, USA, pp 672679.
  • 22
    U.S. Environmental Protection Agency. 1993. Determination of nitrate-nitrite by automated colorimetry, EPA Method 353.2 (Revision 2.0). Methods for the determination of inorganic substances in environmental samples. EPA/600/R-93/100. Cincinnati, OH.
  • 23
    U.S. Environmental Protection Agency. 1993. Determination of ammonia nitrogen by semi-automated colorimetry, EPA Method 350.1 (Revision 2.0). Methods for the determination of inorganic substances in environmental samples. EPA/600/R-93/100. Cincinnati, OH.
  • 24
    Banks MK, Schwab AP, Cofield N, Alleman JE, Switzenbaum M, Shalabi J, Williams P. 2006. Biosolids-amended soils: Part I. Effect of biosolids application on soil quality and ecotoxicity. Water Environ Res 78: 22172230.
  • 25
    Lawrence AP, Bowers MA. 2002. A test of the hot mustard extraction method of sampling earthworms. Soil Biol Biochem 34: 549552.
  • 26
    Higgins CP, Paesani ZJ, Abbott Chalew TA, Halden RU. 2009. Bioaccumulation of triclocarban in lumbriculus variegatus. Environ Toxicol Chem 28: 25802586.
  • 27
    Snyder EH, O'Connor GA, McAvoy D. 2011. Toxicity and bioaccumulation of biosolids-borne triclocarban (TCC) in terrestrial organisms. Chemosphere 82: 460467.
  • 28
    Shareef A, Angove MJ, Wells JD. 2006. Optimization of silyation of using N-methyl-N-(trimethylsilyl)-trifluoroacetamide, N,O-bis (trimethylsilyl)-trifluoro acetamide and N-(tert-butyldemethylsilyl)-N-methyltrifluoroacetamide for the determination of the estrogens, estrone and 17 alpha-ethinylestradiol by gas chromatography-mass spectrometry. J Chromatogr A 1108: 121128.
  • 29
    SAS Institute. 2002. SAS 9.1.3 Documentation. Cary, NC, USA.
  • 30
    Barbarick KA, Doxtader KG, Redente EF, Brobst RB. 2004. Biosolids effects on microbial activity in shrub land and grassland soils. Soil Sci 169: 176187.
  • 31
    Sullivan TS, Stromberger ME, Paschke MW, Ippolito JA. 2006. Long-term impacts of infrequent biosolids applications on chemical and microbial properties of a semi-arid rangeland soil. Biol Fertil Soil 42: 258266.
  • 32
    Holt LM, Laursen AE, McCarthy LH, Vadim Bostan I, Spongberg AL. 2010. Effects of land application of municipal biosolids on nitrogen-fixing bacteria in agricultural soil. Biol Fertil Soil 46: 407441.
  • 33
    Franco-Hernandez O, Dendooven L. 2006. Dynamics of C, N, and P in soil amended with biosolids from a pharmaceutical industry producing cephalosporines or third generation antibiotics: A laboratory study. Bioresource Technol 97: 15631571.
  • 34
    Peijnenburg WJGM, Vijver MG. 2009. Earthworms and their use in eco (toxico) logical modeling. In Devillers J, ed, Ecotoxicol Modeling. Springer, NY, USA, pp 177204.
  • 35
    Morrison HA, Gobas FAPC, Lazar R, Haffner GD. 1996. Development and verification of a bioaccumulation model for organic contaminants in benthic invertebrates. Environ Sci Technol 30: 33773384.
  • 36
    Trent University. 2009. Biosolids Amended Soil Level IV (BASL-4), v1.10. Trent University, Peterborough, Ontario.
  • 37
    Hendriks AJ, Ma WC, Brouns JJ, de Ruiter-Dijkman EM, Gast R. 1995. Modeling and monitoring organochlorine and heavy metal accumulation in soils, earthworms, and shrews in Rhine-Delta floodplains. Arch Environ Contam Toxicol 29: 115127.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

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