Ecotoxicological Effects of Aflatoxins on Earthworms under Different Temperature and Moisture Conditions.

Aflatoxin contamination remains one of the most important threats to food safety and human health. Aflatoxins are mainly found in soil, decaying plant material and food storage systems and are particularly abundant during drought stress. Regulations suggest the disposal of aflatoxin-contaminated crops by incorporation into the soil for natural degradation. However, the fate and consequences of aflatoxin in soil and on soil organisms providing essential ecological services remain unclear and could potentially pose a risk to soil health and productivity. The protection of soil biodiversity and ecosystem services are essential for the success of the declared United Nations Decade on Ecosystem Restoration. The focus of this study was to investigate the toxicological consequences of aflatoxins to earthworms' survival, growth, reproduction and genotoxicity under different temperature and moisture conditions. Results indicated an insignificant effect of aflatoxin concentrations between 10 and 100 µg/kg on the survival, growth and reproduction but indicated a concentration-dependent increase in DNA damage at standard testing conditions. However, the interaction of the toxin with different environmental conditions, particularly low moisture, resulted in significantly reduced reproduction rates and increased DNA damage in earthworms.

Do Endemic Soil Fauna Species Deserve Extra Protection for Adverse Heavy Metal Conditions?

The concept of Ecotoxicological Species Sensitivity Distributions, as used in EU and US, to derive environmental standards for contaminants, starts from the assumption that by protecting the majority of species (95% confidence interval) all species will be protected. Nevertheless, 5% of the species outside the confidence interval might become harmed; half of it being the most sensitive for the particular compound tested. With respect to protection of rare endemic species it is not clear, however, if contamination is a driving factor for endemicity. The aim of this paper is to explore whether endemic and rare species deserve extra protection from adverse environmental conditions. To this end, a brief overview of the various forms of endemism, their relation to environmental stress factors and the distribution of endemic species is discussed. Further, the sensitivities of these species towards environmental stress factors are analysed, in order to conclude if and how endemic species could be better protected against environmental stress factors. This was achieved by specifically focusing on the potential impacts of metalliferous soils, mining, the treatment of mined soil and the storage of treated mine waste. It is concluded that at present there are some signals about specific sensitivities, but the database is much too small for a definite conclusion about adverse environmental factors as a threat to endemic species. The data gap has to be filled in with experimental tests with endemic species. This is hampered by the protection status of these endemic, rare species. Recommendations and derived activities are proposed to address this.

Global Trends of Green Pesticide Research from 1994 to 2019: A Bibliometric Analysis.

The fast-growing world population places food production under enormous pressure to ensure food security. One of the most common methods to increase food production is the use of pesticides, but the continuous use thereof has numerous detrimental effects on the environment. The interest in biopesticides for a possible substitute has grown over the past two decades. To determine the research evolution of biopesticides (green pesticides), a bibliometric analysis from 1994 to 2019 was carried out. A total of 580 documents were found eligible in the Scopus database for this analysis. Parameters such as the number of articles, article citations, keywords, source impact, and countries of publication were used to analyse the documents and rank countries based on authors, productivity, article citations, and co-authorship. The analysis reveals production increased significantly from 2009 and has the most published documents in 2019 with a total of 74 articles. Asia's most populous countries, India and China, were ranked first and second, respectively, and the USA third in terms of the most productive countries in the field of plant biopesticides. Countries in Europe and Africa however have fewer publications than expected in this field, given the fact that they are high consumers of pesticides. India, China, and the USA have 4.08%, 2.94%, and 12.5% multiple country publications (MCPs), respectively, with the USA having a stronger collaboration. Finally, there is a clear indication in this study that India and China are taking the lead in substituting synthetic pesticides with the alternative natural plant biopesticide.

Aflatoxins in the soil ecosystem: an overview of its occurrence, fate, effects and future perspectives.

Aflatoxins are secondary metabolites produced by specific strains of fungi, especially Aspergillus spp. These natural toxins are mainly found in soil, decaying vegetation and food storage systems and are particularly abundant during drought stress. Aflatoxin contamination is one of the most important threats to food safety and human health due to its toxic, mutagenic and carcinogenic properties. Therefore, most research focuses on post-harvest contamination of aflatoxins in feed and food commodities but very limited information is available about aflatoxin contamination and its toxicological consequences in the soil ecosystem. Current regulations provide minimal options for the disposal of aflatoxin-contaminated crops, amongst which is the incorporation of residues into the soil for natural degradation. This form of mycotoxin loading into the soil could potentially change its physicochemical characteristics and biotic parameters. Recent studies suggest that as climate conditions change, the occurrence and geographical distribution of aflatoxins might increase, posing significant health risks to the soil ecosystem, food crop production and human health. This review will focus on studies that look at the environmental and toxicological consequences of aflatoxin contamination with the aim of clarifying the risk that aflatoxin contamination poses to soil ecosystems. Many aspects of aflatoxin occurrence, degradation and the effects of its transformation products in the soil environment are still unknown and remain an important area of research for soil health and productivity. A climatic approach, in terms of changes in soil moisture and air temperature, is important for future risk assessments of aflatoxin contamination.

Structural and functional differentiation of bacterial communities in post-coal mining reclamation soils of South Africa: bioindicators of soil ecosystem restoration.

Soil microbial communities are suitable soil ecosystem health indicators due to their sensitivity to management practices and role in soil ecosystem processes. Presently, information on structural and functional differentiation of bacterial communities in post-coal mining reclamation soils of South Africa is sparse. Here, bacterial communities in three post-coal mining reclamation soils were investigated using community-level physiological profiling (CLPP), enzyme activities, and next-generation sequencing of 16S rRNA gene. Inferences were drawn in reference to adjacent unmined soils. CLPP-based species diversity and proportionality did not differ significantly (P > 0.05) whereas activities of ß-glucosidase, urease and phosphatases were significantly (P < 0.05) influenced by site and soil history (reclaimed vs unmined). Bacterial communities were influenced (PERMANOVA, P < 0.05) by soil history and site differences, with several phylotypes differentially abundant between soils. Contrastingly, predicted functional capabilities of bacterial communities were not different (PERMANOVA, P > 0.05), suggesting redundancy in bacterial community functions between reclamation and unmined soils. Silt content, bulk density, pH, electrical conductivity, Na and Ca significantly influenced soil bacterial communities. Overall, results indicate that bacterial community structure reflects underlying differences between soil ecosystems, and suggest the restoration of bacterial diversity and functions over chronological age in reclamation soils.

Heavy Metal Threats to Plants and Soil Life in Southern Africa: Present Knowledge and Consequences for Ecological Risk Assessment.

In recent times there has been remarkable development in the field of soil ecotoxicology and risk assessment (RA) models. It is, however, debatable if these RA models are robust representatives for worldwide relevance. In order to investigate this, the current overview aims to address heavy metal threats to soil life in southern Africa by investigating present knowledge and consequences for RA using research in southern Africa as a case. To this end, the focus is on southern African soils, soil life and living conditions. To critically discuss these issues, we report on extensive research conducted in the southern African context and looked how comparable these findings are to RA models employed in the western world. This is done by providing an inventory of selected studies focused on the ecotoxicity of metals towards soil life. It is concluded that there is a dearth of information on southern African soil life, most of which are laboratory-based studies carried out by a handful of researchers. Future research incorporating the available information into a soil ecosystem assessment procedure is paramount. It is recommended that a starting point to tackle this might be the development of holistic sight-specific guidelines for ecological risk assessment at larger spatial scales (km2) which takes into cognizance landscapes, vegetation and faunal characteristics.

Effect of Bacillus cereus on the ecotoxicity of metal-based fungicide spiked soils: Earthworm bioassay.

Soil microorganisms exhibit varying levels of metal tolerance across a diverse range of environmental conditions. The use of metal-based fungicides such as mancozeb and copper oxychloride could potentially result in increased levels of manganese, zinc and copper which may adversely affect soil mesofauna. Under standardized earthworm bioassay conditions (temperature, pH, soil type and water content), we investigated the effect of Bacillus cereus on mancozeb and copper oxychloride ecotoxicity towards Eisenia andrei. A metal-tolerant Bacillus cereus strain previously isolated from a gold mining site was introduced into fungicide spiked soils. Earthworms were exposed to bacterial inoculated and non-inoculated substrates of mancozeb (8, 44, 800 and 1250 mg kg-1) and copper oxychloride (200, 450, 675 and 1000 mg kg-1). Experimental trials assessed avoidance-behavior, growth and reproduction utilizing standardized protocols (ISO and OECD). In the avoidance-behavior, E. andrei showed significant (p< 0.05) preference for inoculated substrates. Further, significant (p< 0.05) increases in biomass, survival, cocoons, juveniles and lower soil and tissue Mn, Cu and Zn contents were recorded at 8 and 44 mg kg-1 mancozeb and copper oxychloride 200 and 450 mg kg-1 inoculated soils compared to non-inoculated. However, at 800 and 1250 mg kg-1 mancozeb and 675 and 1000 mg kg-1 copper oxychloride concentrations, reproductive success in both inoculated and non-inoculated treatments was negatively (p< 0.05) affected. In conclusion, Bacillus cereus decreased the ecotoxicity of metal-based fungicides towards Eisenia andrei at 8 and 44 mg kg-1 mancozeb and 200 and 450 mg kg-1 copper oxychloride concentrations. The outcome observed with the inoculated substrates at elevated fungicides concentrations maybe as a result of the environmental conditions (pH and temperature).

Sublethal Effects of Ionic and Nanogold on the Nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans is used as an ecotoxicological model species in both aqueous medium and solid substrates. It is easy and of low cost to maintain in the laboratory and it produces hundreds of offspring within a short period of time. It also has a small body size (1 mm), making it possible for in vivo assays to be conducted in 12-well plates. Engineered nanomaterials (ENMs) are a class of emerging pollutants. Nanogold (nAu) is used in many consumer products and in vivo drug delivery. These materials can be released into the aquatic environment during production or discarding of consumer products. As nAu is insoluble in water, the sediment would become the final depository for the materials. It has become increasingly important to use sediment dwelling organisms to screen for possible toxicity of these ENMs. In this study C. elegans was exposed to a range of concentrations of nAu and ionic gold in M9-media, acting as a substitute for pore water. After 96-hour growth, fertility and reproduction were determined. Internal structure damage and internalisation of particles in C. elegans were determined by using SEM and CytoViva® Darkfield Imaging. From these images the nanomaterials are distributed around the oocytes in the reproductive organs, as well as the pharynx. Results obtained indicate that nAu affects reproduction more than growth due to internal gonad damage, albeit at very high exposure concentrations, indicating no toxicity at environmentally relevant concentrations. Ionic Au is more toxic than nAu and effects fertility and reproduction due to ion release. These results give more information regarding the toxicity and in vivo uptake of nAu and form part of an environmental risk assessment of ENMs.

Heavy metal tolerance traits of filamentous fungi isolated from gold and gemstone mining sites

ABSTRACT Increased environmental pollution has necessitated the need for eco-friendly clean-up strategies. Filamentous fungal species from gold and gemstone mine site soils were isolated, identified and assessed for their tolerance to varied heavy metal concentrations of cadmium (Cd), copper (Cu), lead (Pb), arsenic (As) and iron (Fe). The identities of the fungal strains were determined based on the internal transcribed spacer 1 and 2 (ITS 1 and ITS 2) regions. Mycelia growth of the fungal strains were subjected to a range of (0-100 Cd), (0-1000 Cu), (0-400 Pb), (0-500 As) and (0-800 Fe) concentrations (mgkg-1) incorporated into malt extract agar (MEA) in triplicates. Fungal radial growths were recorded every three days over a 13-days' incubation period. Fungal strains were identified as Fomitopsis meliae, Trichoderma ghanense and Rhizopus microsporus. All test fungal exhibited tolerance to Cu, Pb, and Fe at all test concentrations (400-1000 mgkg-1), not differing significantly (p > 0.05) from the controls and with tolerance index >1. T. ghanense and R. microsporus demonstrated exceptional capacity for Cd and As concentrations, while showing no significant (p > 0.05) difference compared to the controls and with a tolerance index >1 at 25 mgkg-1 Cd and 125 mgkg-1 As. Remarkably, these fungal strains showed tolerance to metal concentrations exceeding globally permissible limits for contaminated soils. It is envisaged that this metal tolerance trait exhibited by these fungal strains may indicate their potentials as effective agents for bioremediative clean-up of heavy metal polluted environments.

Tolerance and growth kinetics of bacteria isolated from gold and gemstone mining sites in response to heavy metal concentrations.

Response and growth kinetics of microbes in contaminated medium are useful indices for the screening and selection of tolerant species for eco-friendly bio-augmentative remediation of polluted environments. In this study, the heavy metal (HM) tolerance, bioaccumulation and growth kinetics of seven bacterial strains isolated from mining sites to 10 HMs (Cd, Hg, Ni, Al, Cr, Pb, Cu, Fe, Mn and Zn) at varied concentrations (25-600 mgL-1) were investigated. The isolates were phylogenetically (16S rRNA gene) related to Lysinibacillus macroides, Achromobacter spanius, Bacillus kochii, B. cereus, Klebsiella pneumoniae, Pseudomonas mosselii and P. nitroreducens. Metal tolerance, effects on lag phase duration and growth rates were assessed using the 96-well micro-titre method. Furthermore, metal bioaccumulation and quantities within cells were determined by transmission electron microscopy and electron dispersive x-ray analyses. Tolerance to Ni, Pb, Fe and Mn occurred at highest concentrations tested. Growth rates increased with increasing Fe concentrations, but reduced significantly (p < .05) with increasing Zn, Cu, Hg, Cd and Al. Significantly higher (p < .05) growth rates (compared to controls) was found with some isolates in Hg (25 mgL-1), Ni (100 mgL-1), Cr (150 mgL-1), Mn (600 mgL-1), Pb (100 mgL-1), Fe (600 mgL-1) and Al (50 mgL-1). Lag phase urations were isolate- and heavy metal-specific, in direct proportion to concentrations. A. spanius accumulated the most Mn and Zn, while B. cereus accumulated the most Cu. Metals accumulated intra-cellularly without cell morphology distortions. The isolates' multi-metal tolerance, intra-cellular metal bioaccumulation and growth kinetics suggest potentials for application in the synergetic biodegradation and bioremediation of polluted environments, especially HM-rich sites.

Heavy metal tolerance traits of filamentous fungi isolated from gold and gemstone mining sites.

Increased environmental pollution has necessitated the need for eco-friendly clean-up strategies. Filamentous fungal species from gold and gemstone mine site soils were isolated, identified and assessed for their tolerance to varied heavy metal concentrations of cadmium (Cd), copper (Cu), lead (Pb), arsenic (As) and iron (Fe). The identities of the fungal strains were determined based on the internal transcribed spacer 1 and 2 (ITS 1 and ITS 2) regions. Mycelia growth of the fungal strains were subjected to a range of (0-100 Cd), (0-1000 Cu), (0-400 Pb), (0-500 As) and (0-800 Fe) concentrations (mgkg-1) incorporated into malt extract agar (MEA) in triplicates. Fungal radial growths were recorded every three days over a 13-days' incubation period. Fungal strains were identified as Fomitopsis meliae, Trichoderma ghanense and Rhizopus microsporus. All test fungal exhibited tolerance to Cu, Pb, and Fe at all test concentrations (400-1000mgkg-1), not differing significantly (p>0.05) from the controls and with tolerance index >1. T. ghanense and R. microsporus demonstrated exceptional capacity for Cd and As concentrations, while showing no significant (p>0.05) difference compared to the controls and with a tolerance index >1 at 25mgkg-1 Cd and 125mgkg-1 As. Remarkably, these fungal strains showed tolerance to metal concentrations exceeding globally permissible limits for contaminated soils. It is envisaged that this metal tolerance trait exhibited by these fungal strains may indicate their potentials as effective agents for bioremediative clean-up of heavy metal polluted environments.

Amelioration of iron mine soils with biosolids: Effects on plant tissue metal content and earthworms.

The achievement of environmentally sound and economically feasible disposal strategies for biosolids is a major issue in the wastewater treatment industry around the world, including Swaziland. Currently, an iron ore mine site, which is located within a wildlife sanctuary, is being considered as a suitable place where controlled disposal of biosolids may be practiced. Therefore, this study was conducted to investigate the effects of urban biosolids on iron mine soils with regard to plant metal content and ecotoxicological effects on earthworms. This was done through chemical analysis of plants grown in biosolid-amended mine soil. Earthworm behaviour, reproduction and bioaccumulation tests were also conducted on biosolid-amended mine soil. According to the results obtained, the use of biosolids led to creation of soil conditions that were generally favourable to earthworms. However, plants were found to have accumulated Zn up to 346 mg kg-1 (in shoots) and 462 mg kg-1 (in roots). This was more than double the normal Zn content of plants. It was concluded that while biosolids can be beneficial to mine soils and earthworms, they can also lead to elevated metal content in plant tissues, which might be a concern to plant-dependant wildlife species. Nonetheless, it was not possible to satisfactorily estimate risks to forage quality since animal feeding tests with hyperaccumulator plants have not been reported. Quite possibly, there may be no cause for alarm since the uptake of metals from soil is greater in plants grown in pots in the greenhouse than from the same soil in the field since pot studies fail to mimic field conditions where the soil is heterogeneous and where the root system possesses a complex topology. It was thought that further field trials might assist in arriving at more satisfactory conclusions.

Preliminary evidence of differences in cadmium tolerance in metal-free stocks of the standard earthworm test species Eisenia andrei (Oligochaeta).

To test whether metal-tolerant and metal-sensitive earthworm specimens could be an inherent part of metal-free earthworm populations, (i) we used DNA barcoding to identify and categorize earthworms from 8 populations of the standard test species Eisenia andrei, and (ii) the earthworms carrying three of the identified COI haplotypes (named Hap1, hap3 and Hap3) were paired up and exposed to Cd in order to assess the difference in Cd sensitivity between the breeding pairs. A total of six breeding pairs were exposed to 0, 25, 50 and 100 mg Cd/kg for 4 weeks at 20 °C. The survival of the breeding pairs, their change in biomass and cocoon production were assessed. For all of the endpoints assessed, the results indicated that couple 6 (Hap3 × Hap3) was the most sensitive breeding pair whereas couple 4 (Hap1 × Hap3) was the least sensitive one. The analysis of Cd tissue contents revealed that with increasing Cd concentration, Cp6 (Hap3 × Hap3) could accumulate significantly more Cd than any other breeding pair (p ≤ 0.01). Our findings indicate that E. andrei may harbour intrinsically Cd-tolerant and Cd-sensitive individuals and that this may be due to individual differences in Cd accumulation kinetics. In the context of ecotoxicological testing, our results underline the importance of using genetically diverse populations in laboratory testing to prevent generating flawed data from genetically homogeneous laboratory stocks. Although we do not regard the present mitochondrial haplotypes as proxy for possibly nuclear encoded traits, we discuss the necessity of a standardised earthworm barcoding protocol that could help not only to confirm the taxonomy of laboratory earthworm stocks but also to select genetically diverse stocks suitable for laboratory testing.

A greenhouse trial to investigate the ameliorative properties of biosolids and plants on physicochemical conditions of iron ore tailings: Implications for an iron ore mine site remediation.

An iron ore mine site in Swaziland is currently (2015) in a derelict state as a consequence of past (1964-1988) and present (2011 - current) iron ore mining operations. In order to control problems associated with mine wastes, the Swaziland Water Services Corporation (SWSC) recently (2013) proposed the application of biosolids in sites degraded by mining operations. It is thought that this practice could generally improve soil conditions and enhance plant reestablishment. More importantly, the SWSC foresees this as a potential solution to the biosolids disposal problems. In order to investigate the effects of biosolids and plants in soil physicochemical conditions of iron mine soils, we conducted two plant growth trials. Trial 1 consisted of tailings that received biosolids and topsoil (TUSB mix) while in trial 2, tailings received biosolids only (TB mix). In the two trials, the application rates of 0 (control), 10, 25, 50, 75 and 100 t ha(-1) were used. After 30 days of equilibration, 25 seeds of Cynodon dactylon were sown in each pot and thinned to 10 plants after 4 weeks. Plants were watered twice weekly and remained under greenhouse conditions for 12 weeks, subsequent to which soils were subjected to chemical analysis. According to the results obtained, there were significant improvements in soil parameters related to fertility such as organic matter (OM), water holding capacity (WHC), cation exchange capacity (CEC), ammonium [Formula: see text] , magnesium (Mg(2+)), calcium (Ca(2+)) and phosphorus ( [Formula: see text] ). With regard to heavy metals, biosolids led to significant increases in soil total concentrations of Cu, Zn, Cd, Hg and Pb. The higher concentrations of Zn and Cu in treated tailings compared to undisturbed adjacent soils are a cause for concern because in the field, this might work against the broader objectives of mine soil remediation, which include the recolonization of reclaimed sites by soil-dwelling organisms. Therefore, while biosolids contain important nutrients that may greatly improve physicochemical conditions and enhance vegetation reestablishment in mined soils, the threat of the build-up of higher levels of trace elements in treated tailings compared to surrounding adjacent soils must not be underestimated.

Utilizing an earthworm bioassay (Eisenia andrei) to assess a South African soil screening value with regards to effects from a copper manufacturing industry.

Metal contamination of soil due to industrialization has become an increasingly important problem in South Africa. This study aimed to investigate the potential impact of a copper (CuSO4·5H2O) production company on the soil environment. Bioassays using Eisenia andrei were performed to assess changes in biomass, reproduction and a biomarker, neutral red retention time, over a 28 day period. Earthworms exposed to soils from the Cu production site differed significantly (p < 0.05) from those exposed to soils 500 m and 5 km away in terms of the measured endpoints. These findings are consistent with the results from the chemical analysis which showed an elevated soil Cu content for both sites closest to the chemical production company compared to the reference site. The results confirm the importance and predictive value of using bioassays in conjunction with chemical analysis during soil quality assessments.

The enchytraeid reproduction test (ERT): A potentially quick and affordable tool for the assessment of metal contaminated soils in emerging economies.

The enchytraeid reproduction test (ERT) was used to assess the ecotoxicity of selected mine tailings and agricultural soils from South Africa. The mine tailings had higher cumulative metal concentrations than agricultural soils. The most contaminated mine tailings significantly reduced the survival of the oligochaete Enchytraeus doerjesi whose reproduction was suppressed in all mine waste substrates. Because it reliably singled out the most contaminated substrate and was found easy to perform, we suggest that the ERT could be a quick and affordable tool for assigning intervention values for soil remediation in emerging economies such as South Africa.

Utilizing Eisenia andrei to assess the ecotoxicity of platinum mine tailings disposal facilities.

South Africa is an important platinum mining country which results in environmental impacts due to the construction of tailing disposal facilities (TDFs). It is unclear what the effects of ageing are on the ecotoxicity of TDFs and whether it increases or decreases over time. The aim of this study was to determine the ecotoxicity of differently aged TDFs by investigating earthworm (Eisenia andrei) responses viz. growth, reproduction, neutral red retention times (NRRT) and tissue metal concentrations. Further, to evaluate the status of these in terms of a geoaccumulation index (I(geo)), pollution index and integrated pollution index. Results indicated that earthworms showed reduced reproductive success (hatchlings per cocoon) and decreased NRRT in all the sites. Juveniles per cocoon between all of the different treatment groups were; control (2.83 ± 0.54) > site 2 (20 years old; 1.83 ± 0.27) > sites 1 and 3 (40 years old; 1.06 ± 0.15 and 6 years old; 0.88 ± 0.39). This might be ascribed to the elevated levels of Cr (±200 to 1,166 µg g(-1)) and Ni (±100 to 316 µg g(-1)) in all of the sites. Earthworms did not bioaccumulate metals with bioconcentration factors for all the different treatments <0.01. Studies like these could be useful when establishing a ranking of TDFs in the future to provide legislative institutions with an indication of the environmental liabilities of platinum mines.

Effects of Cry1Ab transgenic maize on lifecycle and biomarker responses of the earthworm, Eisenia andrei.

A 28-day study was conducted to determine the effects of the Bacillus thuringiensis Cry1Ab toxin on the earthworm Eisenia andrei. Previously, investigations have been limited to life-cycle level effects of this protein on earthworms, and mostly on E. fetida. In this study several endpoints were compared which included biomass changes, cocoon production, hatching success, a cellular metal-stress biomarker (Neutral Red Retention Time; NRRT) and potential genotoxic effects in terms of Randomly Amplified Polymorphic DNA sequences (RAPDs). NRRT results indicated no differences between treatments (p > 0.36), and NRRT remained the same for both treatments at different times during the experiment (p = 0.18). Likewise, no significant differences were found for cocoon production (p = 0.32) or hatching success (p = 0.29). Conversely, biomass data indicated a significant difference between the control treatment and the Bt treatment from the second week onwards (p < 0.001), with the Bt treatment losing significantly more weight than the isoline treatment. Possible confounding factors were identified that might have affected the differences in weight loss between groups. From the RAPD profiles no conclusive data were obtained that could link observed genetic variation to exposure of E. andrei to Cry1Ab proteins produced by Bt maize.