Removal of polycyclic aromatic hydrocarbons from soil amended with biosolid or vermicompost in the presence of earthworms (Eisenia fetida)

Earthworms burrow through the soil thereby accumulating many lipophilic organic pollutants from the surrounding environment, so they could be used to remove polycyclic aromatic hydrocarbons (PAHs) from soil. Sterilized and unsterilized soil was contaminated with phenanthrene (Phen), anthracene (Anth) and benzo[a]pyrene (BaP), with or without added Eisenia fetida and biosolid or vermicompost. Concentrations of PAHs were monitored in soil and earthworms for 70 days. Removal of PAHs increased in soil with earthworms added as 91% of Anth, 16% BaP and 99% Phen was dissipated compared to 42%, 3% and 95% in unamended soil. The microorganisms in the gut of the earthworm contributed to PAHs removal and 100% of Phen, 63% of Anth and 58% of BaP was removed from sterilized soil with E. fetida added. Biosolid and to lesser extent vermicompost accelerated removal of PAHs from soil. Applying earthworms to a contaminated site might be an environmentally friendly way to remove hydrocarbons from soil. However, a limitation might be the cost of the large amounts of earthworms required to remove PAHs from soil and the necessity to supply them with sufficient substrate while maintaining the water content of the soil high enough for their normal functioning.     

Emission of nitrous oxide from hydrocarbon contaminated soil amended with waste water sludge and earthworms

Soils in Mexico are often contaminated with hydrocarbons and addition of waste water sludge and earthworms accelerates their removal. However, little is known how contamination and subsequent bioremediation affects emissions of N₂O and CO₂. A laboratory study was done to investigate the effect of waste water sludge and the earthworm Eisenia fetida on emission of N₂O and CO₂ in a sandy loam soil contaminated with the polycyclic aromatic hydrocarbons (PAHs): phenanthrene, anthracene and benzo(a)pyrene. Emissions of N₂O and CO₂, and concentrations of inorganic N (ammonium (NH₄⁺), nitrite (NO₂^?) nitrate (NO₃^?)) were monitored after 0, 5, 24, 72 and 168 h. Adding E. fetida to the PAHs contaminated soil increased CO₂ production rate significantly 2.0 times independent of the addition of sludge. The N₂O emission rate from unamended soil expressed on a daily base was 5 μg N kg^?1 d^?1 for the first 2 h and increased to a maximum of 325 μg N kg^?1 d^?1 after 48 h and then decreased to 10 μg N kg^?1 d^?1 after 168 h. Addition of PAHs, E. fetida or PAHs + E. fetida had no significant effect on the N₂O emission rate. Adding sludge to the soil sharply increased the N₂O emission rate to >400 μg N kg^?1 d^?1 for the entire incubation with a maximum of 1134 μg N kg^?1 d^?1 after 48 h. Addition of E. fetida, PAHs or PAHs + E. fetida to the sludge-amended soil reduced the N₂O emission rate significantly compared to soil amended with sludge after 24 h. It was found that contaminating soil with PAHs and adding earthworms had no effect on emissions of N₂O. Emission of N₂O, however, increased in sludge-amended soil, but addition of earthworms to this soil and contamination reduced it.     

Comparisons of metal accumulation and excretion kinetics in earthworms (Eisenia fetida) exposed to contaminated field and laboratory soils

The uptake and excretion kinetics of cadmium, copper, lead and zinc were studied for Eisenia fetida exposed to mixtures of these metals in field and OECD artificial soil. Body burdens in worms exposed to all contaminated soils increased over the duration of the experiment. Highest accumulation rates were for worms exposed to the most polluted soils. Pronounced differences were found in the uptake and excretion patterns for essential and non-essential elements (particularly in field soils). For cadmium and lead (non-essential), an equilibrium plateau was not reached during the uptake study and slow excretion was found on transfer of worms to clean soil. For copper and zinc (essential), fast initial uptake was followed by equilibrium after only a few days exposure. Rapid excretion was found after transfer to clean soil, with half-lives of less than 1 day for both metals. A previous study of the effects of metals on worms exposed in OECD and field soils had indicated a higher toxicity in the artificial medium. Thus, in the present study, it was anticipated that greater toxicity would be reflected by increased body burdens for worms in OECD soil. This was, however, not the case. Explanations are given that might account for the fact that the greater toxicity in OECD soil is not invariably accompanied by higher metal burdens. These include the presence of high concentrations of very toxic and highly available ions in laboratory tests and potential differences in the importance of soluble and total metal concentration for determining toxicity and body burdens.     

Nitrogen mineralization in soil amended with sewage sludge and fly ash

Summary The effect of fly ash on N mineralization in sewage sludge was studied during a 5-week aerobic incubation of soil-waste mixtures at different loading rates under controlled conditions. Periodically, the mixtures were leached with distilled water and the inorganic N released was determined in the percolates. The data were tested by an analysis of variance with repeated measures. Significant differences were found among different incubation periods and also between different treatments. The net N mineralization, expressed as a percentage of organic N added in the sludge, was drastically reduced when higher rates (500 Mg ha^-1) of fly ash were added.     

Dynamics of phosphorus phytoavailability in soil amended with stabilized sewage sludge materials

Reducing the environmental risk of soluble P loss from sludge-amended soils is essential for increasing soils capacity to utilize sewage sludge beneficially. Fresh dewatered anaerobically digested sewage sludge (FSS), stabilized with ferrous sulphate (FeSul–SS), calcium oxide (CaO–SS) and aluminum sulphate (alum–SS), each at three chemical-to-FSS ratios, or by composting (BSC), was applied to alluvial soil at rates of 150 and 300 mg P kg^? 1 soil. Changes in P phytoavailability in comparison to KH₂PO₄-amended soil were probed during 100 days of incubation by a P-bioassay and were compared to the concentration of water-soluble P (WSP) and Olsen-P. P phytoavailability was notably linked to the incubation duration and the stabilization process. In general, P phytoavailability at equal P-addition rates was KH₂PO₄ > > alum–SS > BSC ≥ FSS > CaO–SS > > FeSul–SS; and it was positively related to the added P rates, although with quite different patterns among the various sludge products. The concentration of inorganic WSP (WSP_i) extracted from the soil increased following the application of FSS or BSC, and additional P mineralization further increased its concentration during incubation. In contrast, in most cases the chemically stabilized sludges, especially the FeSul–SS, showed considerably reduced inorganic WSP concentrations relative to the untreated soil. The total WSP, Olsen-P and organic WSP (WSP_o) positively correlated to P phytoavailability, indicating that WSP_o plays a role in plant P utilization in these soils. It is concluded that all the chemically stabilized sewage sludge studied effectively controlled WSP_i in soil while still supplying P to support plant growth.     

Initial organic matter transformation of soil amended with composted sewage sludge

 An incubation experiment with composted sewage sludge (CSS) just added to the soil was conducted to determine its initial effects on C decomposition, N nitrification and the transformation of organic matter. CSS was mixed with a sandy loam soil from uncultivated ochric epipedon of a Typic Haploxeralf at rates of 0, 40 and 80 t ha^–1 (dry weight). The data obtained showed that with regard to the unamended soil, both the 40 and the 80 t ha^–1 treatments produced the same result in decreasing respiratory activity, but the addition of increasing amounts of CSS progressively delayed C decomposition. The nitrification index (NI), defined as the relation between nitrate-N and nitrate-N + ammonium-N, increased in correlation with the C mineralization coefficient. Total organic matter decreased after incubation whereas the humic substances increased in relation to the total C mineralized. Received: 28 October 1999     

Behaviour of cadmium and nickel in a soil amended with sewage sludge

Various urban and industrial sewage sludges were applied to a soil at two doses (50 and 100 t ha⁻¹ y⁻¹) during eight years in a field experiment. The soil was analysed at two depths (0–30 and 30–60 cm) for extractable cadmium and nickel. In general these trace metal increased with dosage. However, cadmium formed complexes with organic matter and nickel bound to iron and manganese oxides. Hence, the available fractions of these metals constituted a small proportion of the total content. The results obtained show a low risk of contamination due to the available fractions of these metals at sludges dosages of up to 100 t ha⁻¹. Copyright © 2001 John Wiley & Sons, Ltd.     

The effect of earthworms on carbon storage and soil organic matter composition in tropical soil amended with compost and vermicompost

The use of organic matter (OM) amendments is widespread in tropical countries and may be beneficial for soil carbon storage. Interactions between earthworms and OM amendments in tropical soils are largely unknown. The aim of this study was to investigate the effect of bioturbation on the quantity and chemical composition of OM in soil amended with compost and vermicompost. Our approach included comparison of soil samples amended with compost, vermicompost or chemical fertilizers in the presence or absence of earthworms during a one-year greenhouse experiment. The soils were submitted to a regular cultivation cycle. After one year, we analysed bulk samples for soil OM elemental composition and characterised its lignin and non-cellulosic carbohydrate components.Our results showed a decrease of the carbon and nitrogen content in soil amended with chemical fertilizers. Vermicompost amendment led to unchanged OC content, whereas the compost amendment increased the soils OC content compared to initial soil. The addition of earthworms reduced OC and N content in soils with organic amendments. This is in contrast to soil amended with mineral fertilizer only, where the presence of earthworms did not have any effect. Bioturbation influenced the lignin signature of the soils, and to a lesser extent the non-cellulosic carbohydrate signature. In conclusion, compost amendment combined with bioturbation influenced the quality and quantity of SOM and as result carbon storage and its biogeochemical cycling in tropical soils. Implications for soil fertility remain to be elucidated.     

Carbon sequestration in a limestone quarry mine soil amended with sewage sludge

To reclaim a limestone quarry, 200 and 400 Mg/ha of municipal sewage sludge were mixed with an infertile calcareous substrate and spread as mine soil in 1992. Soil samples were taken 1 week later and again after 17 yr of mine soil rehabilitation so as to assess changes in the amount and persistence of soil organic carbon (SOC). Sludge application increased SOC as a function of the sludge rate at both sampling times. Seventeen years after the sludge amendments, the nonhydrolysable carbon was increased in the 400 Mg/ha of sludge treatment. The recalcitrance of SOC was less in sludge‐amended soils than in the control treatment at the initial sampling, but 17 yr later this trend had reversed, showing qualitative changes in soil organic carbon. The CO₂‐C production had not differed between treatments, yet the percentage of mineralized SOC was less in the high sludge dose. When the size of active (C_active) and slow (C_slow) potentially mineralizable C pools was calculated by curve fitting of a double‐exponential equation, the proportion of C_active was observed to be smaller in the 400 Mg/ha sludge treatment. Soil aggregate stability, represented by the mean weight diameter of water‐stable soil aggregates, was significantly greater in mine soil treated with the high dose of sludge (18.5%) and SOC tended to be concentrated in macro‐aggregates (5–2 mm). Results suggest that SOC content in sludge‐amended plots was preserved due by (i) replacement of the labile organic carbon of sludge by more stable compounds and (ii) protection of SOC in aggregates.     

Biological and physical resilience of soil amended with heavy metal-contaminated sewage sludge

Stability and resilience of a variety of soil properties and processes are emerging as key components of soil quality. We applied recently developed measures of biological and physical resilience to soils from an experimental site treated with metal‐contaminated sewage sludge. Soils treated with cadmium‐, copper‐ or zinc‐contaminated, digested or undigested sewage sludge were studied. Biological stability and resilience indices were: (i) the time‐dependent effects of either a transient stress (heating to 40°C for 18 hours) or a persistent stress (amendment with CuSO₄) on decomposition, and (ii) the mineralization of dissolved organic carbon (DOC) released by drying–rewetting cycles. Physical stability and resilience measures were: (i) compression and expansion indices of the soils, and (ii) resistance to prolonged wetting and structural regeneration through drying–rewetting cycles. Soil total carbon and DOC levels were greater in the sludge‐amended soils, but there were no differential effects due to metal contamination of the sewage sludge. Effects of metals on physical resilience were greater than effects on soil C, there being marked reductions in the expansion indices with Cd‐ and Cu‐contaminated sludge, and pointed to changes in soil aggregation. The rate of mineralization of DOC released by drying and wetting was reduced by Zn contamination, while biological resilience was increased in the Zn‐contaminated soil and reduced by Cd contamination. We argue that physical and biological resilience are potentially coupled through the microbial community. This needs to be tested in a wider range of soils, but demonstrates the benefits from a combined approach to the biological and physical resilience of soils.     

Soybean nodulation and nitrogen fixation on soil previously amended with sewage sludge

Summary A greenhouse study was conducted to examine the residual effects of sewage sludge on soybean Glycine max (L.) Merr., nodulation, and N fixation. Nodulating and nonnodulating isolines of Clark soybean were grown to the R2 stage in soils (Typic Paleudults) obtained from plots where heat-treated sludge had been applied in 1976 at rates equal to 0, 56,112, and 224 Mg ha^–1 high (7.0) and low (6.2) soil pH regimes were established by CaCO₃ additions. Sludge and soil pH treatments resulted in clearly defined differences in metal uptake by soybean shoots. Plant Zn, Cd, and Ni concentrations were greater on pH 6.2, sludge-amended soil than on the pH 7.0, amended soil. At low soil pH, soybean Zn and Cd concentrations, respectively, increased from 41 and 0.19 mg kg^–1 (control) to 120 and 0.58 mg kg^–1 at the 224 Mg hat sludge rate. At the high soil pH and 224 hg hat sludge rate, Zn and Cd concentrations were 45 and 0.15 mg kg^–1, respectively.Symbiotic N fixation provided 90% of the total N accumulation. Total N accumulation, shoot N concentration, dry matter, and N fixation by nodulating soybeans exhibited a significant linear increase with sludge rate. Total N accumulation, dry matter, and N fixation were significantly greater at high soil pH. For high and low soil pH, respectively, N fixation increased from 422 and 382 mg N per plant (control) to 614 and 518 mg N per plant at the 224 Mg ha^–1 sludge rate. While soybean nodulation also increased linearly on sludge-amended soil, a significant rate times pH interaction for nodule number indicated that nodulation was less strongly enhanced by sludge at low soil pH.     

Labile and stable pools of organic matter in soil amended with sewage sludge biochar

One of the main advantages of using biochar for agricultural purposes is its ability to store carbon (C) in soil for a long-term. Studies of labile and stable fractions of soil organic matter (SOM) may be a good indicator of the dynamics of biochar in soils. This study evaluated the effects of applying sewage sludge biochar (SSB) in combination with mineral fertilizer on fractions of SOM. To conduct this evaluation, 15 Mg ha^?1 of SSB combined or not with mineral fertilizer (NPK) was applied to the soil in two cropping seasons. Apart from total organic C (TOC), the labile and stable fractions of SOM were also determined. The combined use of SSB and NPK resulted in higher TOC, a 22% to 40% increase compared to the control and to the NPK treatments, respectively. The SSB produced at a lower temperature increased the labile fractions of SOM, especially the microbial biomass C, showing its capacity to supply nutrients in the short-term. The stable pools of SOM are increased after adding SSB produced at a higher temperature. It was concluded that pyrolysis temperature is a key-factor that determines the potential of SSB to accumulate C in labile and stable fractions of SOM.     

Heavy metals in mine soils amended with sewage sludge

In order to reclaim a clay quarry, a topsoil material was mixed with gravelly spoil at different ratios and with various rates of sewage sludge. The influence of three spoil/topsoil ratios (1:1, 2:1 and 3:1) and three sludge rates (40, 80 and 120 t ha⁻¹) on chemical properties of the resulting material was investigated, with emphasis on heavy metal (Fe, Cu, Mn, Ni and Zn) contents. The mixtures topsoil/spoil/sludge were water saturated and incubated for 15 or 30 days in a chamber under controlled conditions. The incubated samples were analysed for pH, total carbon and nitrogen, and total, available, exchangeable and soluble heavy metals. The addition of spoil to the topsoil increased the volume of material available, by utilizing an inert material unsuitable by itself to grow plants. The addition of sewage sludge repaired the disadvantages of the spoil, increasing the pH and the organic matter contents. The total heavy metal contents in the mixtures followed the sequence Fe>>Mn>>Zn, Cu>Ni. All except Cu were within the ranges allowed for agricultural lands. The available heavy metals constituted a small fraction of total contents and decrease with time due to complexation and immobilization processes. The exchangeable and soluble fractions were almost negligible; only small amounts of Mn, Zn and Cu were detected. Therefore, the risk of contamination by heavy metals is insignificant in the conditions investigated. Copyright © 1999 John Wiley & Sons, Ltd.     

Bioremediation of the soil contaminated with petroleum oil products using sewage sludge

ABSTRACT

The remediation technologies of soils contaminated with petroleum products are developed in two main directions: the first one encompasses searching for new effective bioagents and the other one explores the ways to activate those microorganisms present in the soil that are capable of degrading oil. The objective of this research was to determine if it is possible to increase the effectiveness of biodegradation of petroleum products by using chemical additives. The soil was supplemented with additives: CuSO4, MnSO4, KMnO4, H2O2, 5% and 10% chemical industry plants sludge, 5% and 10% Stock Company ‘Klaipedos vanduo’ (SC‘KV’) municipal wastewater treatment plants sludge. The data suggest that all the additives statistically significantly stimulated the degradation of diesel fuel (F = 12.01; p = .001) and black oil (F = 9.93; p = .001) compared with the control. It was determined that diesel fuel was degraded the fastest in samples with KMnO4, where efficiency of degradation was 90%, and 88% efficency in samples with 10% chemical industry plants sludge. Black oil was degraded the best in samples, where KMnO4 was added: efficiency of degradation was up to 63%. In the samples with 10% of sewage sludge from chemical industry plant degradation efficiency was 62%.     

Residual metal reactivity of humic acids extracted,from soil amended with sewage sludge

Copper, Fe, and Mn were used as probes to investigate residual metal reactivity for humic acid (HA) samples extracted from a loam soil, either non-amended or amended with anaerobically digested sewage sludge for 4, 5, 6, or 7 yr at 90 t ha^?1. yr^?1. Irrespective of their origin, the HA complexes significant amounts of metal, in forms stable against intense water-leaching, in the order Fe > Cl > Mn. Sludge-amended soil HA adsorbed and retained Fe in amounts greater than HA extracted fron non-amended soil. Metal adsorption occurred mainly by cation-exchange replacement of metals previousl: bound to HA. Water-stable Fe³⁺-HA complexes prepared in the laboratory were partially stable agains H⁺ and metal ion exchange reactions, whereas Cu²⁺ and Mn²⁺ in laboratory-prepared, water-stabl HA complexes were desorbed almost completely by these two reactions. Electron spin resonance spectra indicated that the laboratory-prepared metal-HA complexes had a chemical composition and molecula structure similar to that of indigenous metal-HA complexes, which were stable against all leachin, and cation-exchange treatments. Although the HA samples showed a maximal metal binding (i.e. saturation) as metal loading of the sludge-amended soil increased, they still exhibited a high residua binding capacity for the three metals used as probes.     

Changes in biological properties and antioxidant capacity of an agricultural soil amended with sewage sludge

The effects of applying sewage sludge (SS) to agricultural soil (at low rate of 22.5, LRS, and at high rate of 45 t ha^?1 dry basis, HRS) were monitored over a 120-d experimental period. Total organic carbon (TOC), water-soluble organic carbon (WSOC), alkali-soluble phenols, basal respiration, specific enzyme activity, dehydrogenase activity (DH-ase), metabolic potential (MP) and FDA-hydrolytic activity (FDA) were strongly increased by both rates of SS applications. In the SS amended soil, about 70% of the organic C added with the material remained at the end of the experiment. Basal respiration increased with increasing SS doses. The specific enzyme activity and the MP indicate an increase in the enzyme activity in soil.

The addition of SS led to higher values than the control of all the tested parameters up to the end of the experimental period. The antioxidant capacity (trolox equivalent antioxidant capacity, TEAC) was influenced by SS addition only when applied at HRS. After 120 days only HRS value of TEAC (5.13 mM g^?1) was higher than control (4.09 mM g^?1). The pattern of TEAC did not enable any link to be established between antioxidant capacity and both alkali-soluble phenols and basal respiration in soil.     

生活污泥对白菜供磷和土壤磷状况的影响

采用好气培养和盆栽试验以探明污泥磷的肥效,降低污泥施用导致的土壤磷累积引起的环境风险。结果表明,单施污泥土壤有效磷含量和白菜吸磷量均显著低于施用磷酸一铵和鸡粪处理;施用污泥后有利于增加白菜生长后期土壤磷酸酶的活性和土壤Olsen-P含量。在白菜等产量条件下,单施污泥处理土壤中Olsen-P残留量显著高于污泥与化肥混施处理;在P2O5施用量为902~70 kg/hm2时,污泥堆肥磷的肥效为磷酸一铵的25%左右。     

Isolation by isoelectric focusing of humic-urease complexes from earthworm (Eisenia fetida)-processed sewage sludges

 Vermicomposting is an eco-biotechnological process that transforms energy-rich and complex organic substances into a stabilized humus-like product. In a laboratory experiment, Eisenia fetida (Sav.) earthworms were employed to process putrescible sewage sludges into a high-value biofertilizer, very rich in urease activity and humic-urease complexes (stabilized extracellular enzymes). Extracellular humic-urease complexes were extracted by a single 24-h extraction at 37  °C using neutral pyrophosphate (0.1 M); then, the extracts were dialysed and characterized by means of an analytical isoelectric focusing technique. This technique gave a multiplicity of humic bands enzymatically active, with isoelectric points ranging from 4.8 to 5.6. The results demonstrated that, after an 18-week incubation period, sewage sludge had undergone a biochemical evolution, which caused a doubling of absolute urease activity and a six-fold increase in specific activity (activity with reference to the humic C fraction). The biochemical evolution of the vermicompost was evaluated also from the sharp decrease in pyrophosphate-extractable C and water-soluble C. Stabilization of organic C during vermicomposting and the activity of humic-urease complexes expressed at low pH values are of extreme importance when organic wastes are used in acid soils for biochemical restoration purposes. Received: 10 June 1999     

Heavy metal uptake by blue grama growing in a degraded semiarid soil amended with sewage sludge

Sewage sludge application to semiarid grassland may represent a beneficial means of utilizing this waste product for restoration of degraded sites. Consequently, dried municipal sewage sludge was applied at three rates (22.5, 45, and 90 Mg ha^–1) to a degraded semiarid grassland soil in order to determine the effects of sludge amendments on forage productivity, soil heavy metal content, and metal uptake by blue grama (Bouteloua gracilis).Soil and plant properties in control and amended plots were measured after 1, 2, and 5 growing seasons.Soil nutrients increased linearly with increased sludge application in the first two growing seasons. Consequently, forage quality and total production of blue grama improved significantly over the unamended control as the tissue levels of N, P, K, and crude protein increased. Cadmium and Pb in the sludge-treated plots did not increase significantly over the control after 1 and 2 growing seasons. Levels of DTPA-extractable soil micronutrients (Cu, Fe, Mn, Zn) increased linearly with increased sludge application rate to soil concentrations recommended for adequate plant growth. Soil N, P, and K concentrations remained higher in the sludge-amended soils after 5 growing seasons, while Cu and Cd increased to slightly above desireable limits as the soil pH decreased to 7.4 and 7.0 in the 45 and 90 Mg ha^–1 treatments, respectively. However, with the exception of Mn which remained within desirable limits, metal concentrations (including Cu and Cd) in blue grama tissue were not significantly different from the control treatment after five growing seasons. Based on soil and plant tissue metal concentrations, it appears that sludge applied at rates between 22.5 and 45 Mg ha^–1 will maintain the most favorable nutrient levels coupled with significant improvements in forage production in this semiarid grassland environment.     

Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil,effects of carbaryl

The aim of this study was to investigate the effects of carbaryl, at different concentrations and exposure times, on the biochemical responses of the earthworm, Eisenia fetida andrei (E. andrei) to: (1) elucidate the mechanisms of action of carbamate compounds; and (2) explore the potential for using these responses as biomarkers to monitor carbamate-contaminated soils or for use in sublethal assays to test chemicals in the laboratory. Thus, earthworms were exposed to increasing concentrations of carbaryl (CA) (12, 25 and 50 mg kg⁻¹ artificial soil) for different periods of time (2, 7 and 14 days) using the standard soil test method. The activities of the following enzymes were measured: catalase (CAT), acetylcholinesterase (AChE), glutathione reductase (GR), glutathione-S-transferase (GST), methoxyresorufin-O-deethylase (MROD) and NADH (NADH Red) and NADPH (NADPH Red) cytochrome reductases activities. Levels of lipid peroxides (LP), peroxidizable lipids (LPI), total glutathione (total GSH) and the percentage of oxidized glutathione (%GSSG) were also determined. Our results showed that cholinesterases are the main target of carbaryl in E. andrei. Carbaryl also inhibited biotransformation enzyme activities but did not induce oxidative stress. Changes were detected in phase I and acetylcholinesterase activities upon exposure to the lowest dose of carbaryl (12 mg kg⁻¹). This demonstrated the sensitivity of these parameters in E. andrei. In addition, discrimination between doses and exposure times was clearer when all the responses were considered rather than a selective choice of biomarkers. This confirms our previous finding that a suite of biochemical responses could be used as a sublethal assay for testing chemicals in the laboratory or for soil contamination surveys.