Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments

In Québec, as observed globally, abnormally high honey bee mortality rates have been reported recently. Several potential contributing factors have been identified, and exposure to pesticides is of increasing concern. In maize fields, foraging bees are exposed to residual concentrations of insecticides such as neonicotinoids used for seed coating. Highly toxic to bees, neonicotinoids are also reported to increase AChE activity in other invertebrates exposed to sub-lethal doses. The purpose of this study was therefore to test if the honey bee’s AChE activity could be altered by neonicotinoid compounds and to explore possible effects of other common products used in maize fields: atrazine and glyphosate. One week prior to pollen shedding, beehives were placed near three different field types: certified organically grown maize, conventionally grown maize or non-cultivated. At the same time, caged bees were exposed to increasing sub-lethal doses of neonicotinoid insecticides (imidacloprid and clothianidin) and herbicides (atrazine and glyphosate) under controlled conditions. While increased AChE activity was found in all fields after 2 weeks of exposure, bees close to conventional maize crops showed values higher than those in both organic maize fields and non-cultivated areas. In caged bees, AChE activity increased in response to neonicotinoids, and a slight decrease was observed by glyphosate. These results are discussed with regard to AChE activity as a potential biomarker of exposure for neonicotinoids.     

Bioaccumulation of glyphosate and its formulation Roundup Ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes

The bioaccumulation potential of glyphosate and the formulation Roundup Ultra, as well as possible effects on biotransformation and antioxidant enzymes in Lumbriculus variegatus were compared by four days exposure to concentrations between 0.05 and 5 mg L⁻¹ pure glyphosate and its formulation. Bioaccumulation was determined using ¹⁴C labeled glyphosate. The bioaccumulation factor (BCF) varied between 1.4 and 5.9 for the different concentrations, and was higher than estimated from log P_ow. Glyphosate and its surfactant POEA caused elevation of biotransformation enzyme soluble glutathione S-transferase at non-toxic concentrations. Membrane bound glutathione S-transferase activity was significantly elevated in Roundup Ultra exposed worms, compared to treatment with equal glyphosate concentrations, but did not significantly differ from the control. Antioxidant enzyme superoxide dismutase was significantly increased by glyphosate but in particular by Roundup Ultra exposure indicating oxidative stress. The results show that the formulation Roundup Ultra is of more ecotoxicological relevance than the glyphosate itself.     

In vivo evaluation of three biomarkers in the mosquitofish (Gambusia yucatana) exposed to pesticides

In this study, the acute toxicity and the in vivo effects of commercial chlorpyrifos, carbofuran and glyphosate formulations on cholinesterase (ChE), glutathione S-transferase (GST) and lactate dehydrogenase (LDH) activities of the mosquitofish (Gambusia yucatana) were investigated. In a first phase of the study, head and muscle ChE were characterized with different substrates (acetylthiocholine iodide, s-butyrylthiocholine iodide and propionylthiocholine iodide) and the selective inhibitors eserine hemisulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284C51), and N,N'-diisopropylphosphorodiamic acid (iso-OMPA). The results obtained suggest that the enzyme present in both head and muscle of G. yucatana is mainly acetylcholinesterase (AChE). Acute toxicity was evaluated by exposing fish to several concentrations of single pesticides and of a mixture of chlorpyrifos/glyphosate. LC50 values were determined after 96 h of exposure, except in the case of carbofuran for which LC50 was calculated after 24 h since almost all the fish died within this period. LC50 values were 0.085 mg/l for chlorpyrifos, 17.79 mg/l for glyphosate, 0.636 mg/l for carbofuran and 0.011 mg/l for the chlorpyrifos/glyphosate mixture. A Toxic Unit approach was used to compare the toxicity of chlorpyrifos and glyphosate when occurring in a mixture with their toxicities as single compounds. Synergistic effects of chlorpyrifos and glyphosate when present in a mixture were found. At the end of each bioassay (24 h for carbofuran, 96 for the other substances/mixture), effects on biomarkers were analyzed. Muscle LDH activity was not altered by any of the three pesticides tested. Gill GST activity was significantly inhibited (40%) by carbofuran after 24 h of exposure to concentrations equal or higher than 0.06 mg/l. ChE muscle and head activity were significantly inhibited (50% and 30%, respectively) by carbofuran at concentrations equal or higher than 0.25 mg/l. Chlorpyrifos induced a significant inhibition of both muscle and head ChE (80% and 50%, respectively) after 96 h of exposure to concentrations equal or higher than 0.05 mg/l. Carbofuran did not induce significant alterations of fish ChE. The ChE EC50 determined for chlorpyrifos/glyphosate mixture (0.070 mg/l) was higher than the correspondent value calculated for chlorpyrifos alone (0.011 mg/l) suggesting an antagonistic effect of glyphosate on ChE inhibition by chlorpyrifos. ChE activity of G. yucatana seems to be a good biomarker to diagnose the exposure of wild populations of this species exposed to anticholinesterase pesticides.     

Toxicity of the herbicide glyphosate to Chordodes nobilii (Gordiida, Nematomorpha)

Nematomorpha (horsehair worms) is a poorly known group of worm-like animals similar to nematodes. Adults are free-living and reproduction takes place in freshwater environments, where preparasitic larvae undergo development. All species have a parasitic juvenil stage and infection may result in the host's death, insects being the most frequent host. Most of the life cycle occurs in freshwater environments, which are often contaminated by different pollutants. Based on the lack of information on the toxicity of herbicides to horsehair worms, the objective of this study is to evaluate the effect of different concentrations of glyphosate (technical grade and formulated product) on Chordodes nobilii (Gordiida, Nematomorpha). Bioassays were performed with embryos and larvae (preparasitic stages), and adults (postparasitic stage). Test organisms were exposed for a short period of time to concentrations ranging between 0.1 and 8 mga.e.l(-1) of glyphosate (technical and formulated). Although embryo development was not inhibited, there was a significant decrease in the infective capacity of larvae derived from eggs that had been exposed to >or= 0.1mg/l. Similar results were obtained for directly exposed larvae. No differences in toxicity were detected between the active ingredient and formulated product. Adult exposed for 96 h to 1.76 mgl(-1) formulated Gly shown a mortality of 50%. Results indicate that C. nobilii is affected at glyphosate concentrations lower than those expected to be found in freshwater environments and those specified in the legislation.     

Effect of airborne bromoxynil-octanoate and metribuzin on non-target plants

The present study was conducted to determine the effects of airborne herbicides on the photosynthesis and growth of non-target plants. Sunflowers at different growth stages were used as test plants and exposed for 24 h in a wind tunnel to a range of concentrations of bromoxynil-octanoate and metribuzin. The quantum yield (phiPSII) and the dry weight were used as response parameters. Results indicate that young sunflower plants are affected by sublethal concentrations of both herbicides, whereas metribuzin proved to be more phytotoxic. Bromoxynil-octanoate and metribuzin concentrations > 0.265 and >0.135 microg/m(3) impair the photosynthetic activity of exposed leaves and concentrations >0.780 and >0.641 microg/m(3) of leaves developed after exposure. Effects on dry weight indicate that younger plants were more susceptible, whereby the response to metribuzin proved to be more dependent on growth stage. Based on these results and considering herbicide concentrations in ambient air, there is a reasonable probability that non-target plants are temporarily at risk of being affected.     

Hormetic effect of glyphosate on Urochloa decumbens plants

Abstract

Urochloa decumbens plants may be reached by herbicide drift from applications of glyphosate from neighboring areas or by variations during applications. Considering the different phenological stages and size of plants in these areas, the amount of active ingredient that reaches the plants probably varies. The objective of this study was to evaluate the effects of the application of different doses of glyphosate on U. decumbens plants. Two greenhouse experiments were conducted with two replications at different times. The first experiment evaluated the biological response of U. decumbens plants to glyphosate doses (0, 2.81, 5.63, 11.25, 22.5, 45, 90, 180, 360, 720, and 1,440?g a.e. ha^?1), with six replications. The second experiment evaluated the response of U. decumbens plants to the application of a selected low dose of 11.25?g a.e. ha^?1. Evaluations of injury were performed at 0, 7, 14, and 21?days after application, and dry weight of plants was determined for each evaluation period. U. decumbens plants increased in dry weight when using the glyphosate dose of 11.25?g a.e. ha^?1. However, plants had different responses to the application of this low dose. It can promote both stimulation and inhibition of plant growth.     

Glyphosate and AMPA of agricultural soil,surface water,groundwater and sediments in areas prevalent with chronic kidney disease of unknown etiology,Sri Lanka

Abstract

Glyphosate, which is commercially available as Roundup®, was the widely used herbicide in Sri Lanka until 2015 and is suspected to be one of the causal factors for Chronic Kidney Disease of unknown etiology (CKDu). This research, therefore, aims at studying the presence of glyphosate and Aminomethylphosphonic acid (AMPA) in different environmental matrices in CKDu prevalent areas. Topsoil samples from agricultural fields, water samples from nearby shallow wells and lakes, and sediment samples from lakes were collected and analyzed for glyphosate and AMPA using the LC/MS. Glyphosate (270–690 µg/kg) and AMPA (2–8 µg/kg) were detected in all soil samples. Amorphous iron oxides and organic matter content of topsoil showed a strong and a moderate positive linear relationship with glyphosate. The glyphosate and inorganic phosphate levels in topsoil had a strong negative significant linear relationship. Presence of high valence cations such as Fe³⁺ and Al³⁺ in topsoil resulted in the formation of glyphosate-metal complexes, thus strong retention of glyphosate in soil. Lower levels of AMPA than the corresponding glyphosate levels in topsoil could be attributed to factors such as the strong adsorption capacity of glyphosate to soil and higher LOQ in the quantification of AMPA. The glyphosate levels of lakes were between 28 to 45 µg/L; no AMPA was detected. While trace levels of glyphosate (1–4 µg/L) were detected in all groundwater samples, AMPA (2–11µg/L) was detected only in four out of nine samples. Glyphosate was detected in all sediment samples (85–1000 µg/kg), and a strong linear relationship with the organic matter content was observed. AMPA was detected (1–15 µg/kg) in seven out of nine sediment samples. It could be inferred that the impact on CKDu by the levels of glyphosate and AMPA detected in the study area is marginal when compared with the MCL of the USEPA (700 µg/L).     

Effects of sub-lethal glyphosate concentrations on growth and photosynthetic performance of non-target species Bolboschoenus maritimus

Glyphosate use has increased over the last decades for the control of invasive plant species in wetland ecosystems. Although glyphosate has been considered ‘environmentally’ safe, its repeated use could increase the toxicological risk derived from diffuse pollution of surface and groundwater on non-target vegetation. A glasshouse study was designed to determine the effect produced by the addition of different sub-lethal doses of glyphosate herbicides (5–30 mg L⁻¹) to the nutrient solution on the growth and photosynthetic apparatus of Bolboschoenus maritimus. Although B. maritimus plants were able to grow and survive after 20 d of exposure to glyphosate, the presence of this herbicide affected their growth, through a direct interaction with the root system. Particularly, at 30 mg L⁻¹ glyphosate, B. maritimus showed ca. 30% of biomass decrease. The reduction in B. maritimus growth was due to a decrease in net photosynthetic rate (A), which ranged between values ca. 11.5 and 5.5 μmol m⁻² s⁻¹ CO₂ for the control and the highest glyphosate treatment, respectively. The response of A to glyphosate could be largely accounted for by non-stomatal limitations, since stomatal conductance was similar in all glyphosate treatments. Thus, A decrease was prompted by the negative impact of herbicide on photochemical (PSII) apparatus, the reduction in the absorption of essential nutrients, the reduction of photosynthetic pigments and possibly the reduction in Rubisco carboxilation capacity. Moreover, glyphosate excess caused photoinhibitory damage. In conclusion, in this study we have shown that herbicide water pollution could be a source of indirect phytotoxicity for B. maritimus.     

Herbicide Roundup® and its main constituents cause oxidative stress and inhibit acetylcholinesterase in liver of Carassius auratus

Roundup® is a glyphosate-based herbicide containing a mixture of surfactants. This paper evaluates the toxic effects of Roundup® and its main constituents on the goldfish, Carassius auratus, after 7 days exposure. Fish were exposed to 0.16, 0.032 and 0.0064 mg/L of Roundup® [containing 41% isopropylamine salt of glyphosate (G.I.S) and 18% polyoxyethylene amine (POEA)], G.I.S, and POEA. Their livers were taken for determining reactive oxygen species (ROS), superoxide dismutase (SOD) activity, malondialdehye (MDA) content and acetylcholinesterase (AChE) activity. Hydroxyl radical (·OH) could be induced by exposing Roundup® at a rate of 43%–111%, G.I.S at 90%–124% and POEA at142%–157%. A decreased SOD activity was observed in fish exposed to G.I.S and POEA. The contents of MDA significantly increased when exposed to Roundup® at all concentrations, 0.16 mg/L G.I.S and 0.032 mg/L POEA. The exposure led to an inhibition of AChE in livers overall during the experimental periods. POEA was more toxic than Roundup® or G.I.S during this experiment. AChE and ·OH are supposed to be sensitive biomarkers of the exposure of Roundup® and its main constituents to C. auratus.     

Earthworms to assess the innocuousness of spent biomixtures employed for glyphosate degradation

In this study, the innocuousness of different biomixtures employed for glyphosate degradation was tested through Eisenia fetida earthworms. Eight biomixtures were prepared with local materials: alfalfa straw (AS), wheat stubble (WS), river waste (RW) and two different soils (A and B). Each biomixture was divided into two equal portions: one without glyphosate application (control substrate) and the other was sprayed with a commercial glyphosate formulation of 1,000 mg glyphosate a.i. kg^?1 biomixture (applied substrate). The bioassay started when all sprayed biomixtures reached high percentages of glyphosate degradation (spent biomixtures). Three parameters were studied: survival, adults and juveniles biomass and reproduction. The results allowed the identification of three biomixtures (AWS, BWS and BWSRW) for good maintenance and development of E. fetida. In addition, at the end of the bioassay two of the viable biomixtures (AWS and BWS) showed the highest performance of juvenile earthworms compared to a reference soil. The Principal Component Analysis (PCA) indicated that the biomixtures containing high silt and clay percentages and minor density renders higher values of earthworm growth and reproduction. Therefore, these innocuous biomixtures can be used as organic amendments or recycled materials for new treatments on biobeds.     

Effects, transfer, and fate of RDX from aged soil in plants and worms

The objectives of this study were to provide data that can be used to predict exposure-based effects of RDX in aged soil on multiple endpoint organisms representing two trophic levels. These data can be used for defining criteria or reference values for environmental management and conducting specific risk assessment. Dose-response experiments formed the basis for the evaluation of toxic effects and transfer of contaminants from soil into two trophic levels. Long-term exposure tests were conducted to evaluate chronic, sublethal, toxicity and transfer of aged soil-based explosives, with RDX as main contaminant. In these tests, plants were exposed for 55 days in the greenhouse, biomass was determined and residues of explosives parent compounds and RDX metabolites were analyzed using HPLC techniques. Worms were exposed for 28 days (Eisenia fetida) and 42 days (Enchytraeus crypticus) in the laboratory, biomass and number were determined, and tissues were analyzed for explosives compounds. The plants tolerated concentrations up to 1,540 mg RDX kg(-1) soil-DW. Biomass of Lolium perenne was not significantly related to soil-RDX concentration, while biomass of Medicago sativa significantly increased. No screening benchmark for RDX in soil for plants was calculated, since concentrations up to 1,540 mg kg(-1) soil failed to reduce biomass by 20% as required for a LOEC. RDX, RDX-metabolite MNX, and accompanying HMX concentrations in plants were significantly related to concentrations in soil after 55 days of exposure (RDX: R(2) = 0.77-0.89; MNX R(2) = 0.53-0.77; HMX: R(2) = 0.67-0.71). The average bioconcentration factors (BCF) were for RDX 17 in L. perenne and 37 in M. sativa, and for HMX 2 in L. perenne and 44 in M. sativa. The worms also tolerated concentrations up to 1,540 mg RDX kg(-1) soil-DW. Biomass of E. fetida adults decreased with soil-RDX concentration, and a LOEC of 1,253 mg kg(-1) soil-DW was estimated. RDX concentrations in E. fetida were significantly related to concentrations in soil after 28-day exposure (R(2) = 0.88). The average BCF in E. fetida for RDX was 1. Because in response to exposure to RDX-contaminated soil the RDX concentrations in plants increased initially and decreased subsequently, while those in worms increased continuously, RDX in worm tissues may accumulate to higher concentrations than in plant tissues, regardless of the low average BCF for worms.     

Effects of low doses of glyphosate on DNA damage,cell proliferation and oxidative stress in the HepG2 cell line

Environmental Science and Pollution Research - We studied the toxic effects of glyphosate in vitro on HepG2 cells exposed for 4 and 24 h to low glyphosate concentrations likely to be...     

Isolation of culturable mycobiota from agricultural soils and determination of tolerance to glyphosate of nontoxigenic Aspergillus section Flavi strains

Glyphosate-based herbicides are extensively used in Argentina's agricultural system to control undesirable weeds. This study was conducted to evaluate the culturable mycobiota [colony forming units (CFU) g^?1 and frequency of fungal genera or species] from an agricultural field exposed to pesticides. In addition, we evaluated the tolerance of A. oryzae and nontoxigenic A. flavus strains to high concentrations (100 to 500 mM – 17,000 to 84,500 ppm) of a glyphosate commercial formulation. The analysis of the mycobiota showed that the frequency of the main fungal genera varied according to the analyzed sampling period. Aspergillus spp. or Aspergillus section Flavi strains were isolated from 20 to 100% of the soil samples. Sterilia spp. were also observed throughout the sampling (50 to 100%). Aspergillus section Flavi tolerance assays showed that all of the tested strains were able to develop at the highest glyphosate concentration tested regardless of the water availability conditions. In general, significant reductions in growth rates were observed with increasing concentrations of the herbicide. However, a complete inhibition of fungal growth was not observed with the concentrations assayed. This study contributes to the knowledge of culturable mycobiota from agricultural soils exposed to pesticides and provides evidence on the effective growth ability of A. oryzae and nontoxigenic A. flavus strains exposed to high glyphosate concentrations in vitro.     

Metal fractionation in soils and assessment of environmental contamination in Vallecamonica, Italy

Metal contamination was investigated in soils of the Vallecamonica, an area in the northern part of the Brescia province (Italy), where ferroalloy industries were active for a century until 2001. The extent in which emissions from ferroalloy plants affected metal concentration in soils is not known in this area. In this study, the geogenic and/or anthropogenic origin of metals in soils were estimated. A modified Community Bureau of Reference sequential chemical extraction method followed by inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses were employed to evaluate the potential bioavailability of Al, Cd, Mn, Fe, Cr, Zn, and Pb in soils. Principal component analysis (PCA) was used to assess the relationships among metal sources in soil samples from different locations. This approach allowed distinguishing of different loadings and mobility of metals in soils collected in different areas. Results showed high concentrations and readily extractability of Mn in the Vallecamonica soils, which may suggest potential bioavailability for organisms and may create an environmental risk and potential health risk of human exposure.     

New bacterial strain of the genus Ochrobactrum with glyphosate-degrading activity

Thirty bacterial strains with various abilities to utilize glyphosate as the sole phosphorus source were isolated from farm soils using the glyphosate enrichment cultivation technique. Among them, a strain showing a remarkable glyphosate-degrading activity was identified by biochemical features and 16S rRNA sequence analysis as Ochrobactrum sp. (GDOS). Herbicide (3 mM) degradation was induced by phosphate starvation, and was completed within 60 h. Aminomethylphosphonic acid was detected in the exhausted medium, suggesting glyphosate oxidoreductase as the enzyme responsible for herbicide breakdown. As it grew even in the presence of glyphosate concentrations as high as 200 mM, Ochrobactrum sp. could be used for bioremediation purposes and treatment of heavily contaminated soils.     

Uptake and effects of the cypermethrin-containing sea lice treatment Excis in the marine mussel,Mytilus edulis

Wild and farmed mussels, Mytilus edulis, coexist with salmon farms in Scottish sea lochs. A synthetic pyrethroid, cypermethrin, is licensed for use on fish farms to control sea lice infestations as a formulation called Excis. In this study, uptake of cypermethrin from Excis exposure is investigated through the use of gas chromatography with mass-spectrometry. The effects of Excis on mussels are also examined by measuring the neutral red retention time of lysosomes, aerial survival and shell closure. The isomeric ratios of cis:trans cypermethrin measured in mussels are around 80:20; a marked increase from 40:60 to which the mussels were exposed. This is most likely due to preferential metabolism of trans-isomers, as the same response is seen in vertebrates. There is a pronounced behavioural effect of shell closure, where mussels exposed to 1000 microg/l cypermethrin shut their shells within an hour of exposure. Arguments are presented for this effect being either a voluntary response on recognition of cypermethrin, or an effect arising from an involuntary action of cypermethrin on the adductor muscle. Even at 1000 microg/l cypermethrin, neutral red retention time and aerial survival are not affected. The data suggest that the responses of mussels shown here are unlikely to occur in the field, even at the concentrations of cypermethrin used in fish cages, for the treatment of sea lice.     

Ultrafine particle emission from incinerators: the role of the fabric filter

Incinerators are claimed to be responsible of particle and gaseous emissions: to this purpose Best Available Techniques (BAT) are used in the flue-gas treatment sections leading to pollutant emission lower than established threshold limit values. As regard particle emission, only a mass-based threshold limit is required by the regulatory authorities. However; in the last years the attention of medical experts moved from coarse and fine particles towards ultrafine particles (UFPs; diameter less than 0.1 microm), mainly emitted by combustion processes. According to toxicological and epidemiological studies, ultrafine particles could represent a risk for health and environment. Therefore, it is necessary to quantify particle emissions from incinerators also to perform an exposure assessment for the human populations living in their surrounding areas. A further topic to be stressed in the UFP emission from incinerators is the particle filtration efficiency as function of different flue-gas treatment sections. In fact, it could be somehow important to know which particle filtration method is able to assure high abatement efficiency also in terms of UFPs. To this purpose, in the present work experimental results in terms of ultrafine particle emissions from several incineration plants are reported. Experimental campaigns were carried out in the period 2007-2010 by measuring UFP number distributions and total concentrations at the stack of five plants through condensation particle counters and mobility particle sizer spectrometers. Average total particle number concentrations ranging from 0.4 x 10(3) to 6.0 x 10(3) particles cm(-3) were measured at the stack of the analyzed plants. Further experimental campaigns were performed to characterize particle levels before the fabric filters in two of the analyzed plants in order to deepen their particle reduction effect; particle concentrations higher than 1 x 10(7) particles cm(-3) were measured, leading to filtration efficiency greater than 99.99%.     

Chitosan/glyphosate formulation as a potential,environmental friendly herbicide with prolonged activity

Although there are many reports on the dangers posed by glyphosate, this herbicide is still one of the most popular and widely used total weed killers. Therefore, great effort should be made to minimize exposure to this herbicide and limit its losses into the environment. The aim of this study was to prepare a new formulation consisted of various molecular weight chitosans with glyphosate and their evaluation toward active substance release, phytotoxicity, and preliminary herbicidal efficiency. The phytotoxicity study of the obtained chitosan/glyphosate formulations was determined based on the Organisation for Economic Co-operation and Development 208 guideline. Among all tested formulations evaluated for phytotoxicity, that containing the highest molecular weight of chitosan was found to be the least toxic, showing simultaneously the most effective herbicidal activity against selected weeds. The release rate of glyphosate from the obtained formulations was dependent on the molecular weight and viscosity of chitosan.     

Leaching of pesticides through normal-tillage and low-tillage soil--a lysimeter study. II. Glyphosate

Glyphosate is a widely used non-selective herbicide. Leaching of glyphosate (N-(phosphonomethyl)glycine) and/or its metabolite AMPA (aminomethylphosphonic acid) was studied in four lysimeters, two of them being replicates from a low-tillage field (lysimeter 3 and 4), the other two being replicates from a normal tillage field (lysimeter 5 and 6). In both cases the soil was a sandy loam soil with 13-14% clay. The lysimeters had a surface area of 0.5 m2 and a depth of 110 cm. Lysimeter 3 and 4 were sprayed with a mixture of 14C-labelled glyphosate and unlabelled glyphosate, while lysimeter 5 and 6 were sprayed with unlabelled glyphosate. The spraying took place September 18, 1997. The total amount of glyphosate sprayed onto each lysimeter was 40 mg, corresponding to 0.8 kg active ingredient per ha. The lysimeters were installed in an outdoor system in Research Centre Flakkebjerg and were thus exposed to normal climatic conditions of the area. A mean of 260 l drainage water were collected from lysimeter 3 and 4 and a mean of 375 litres from lysimeter 5 and 6. The mean yearly concentration of leached glyphosate and/or AMPA was significantly below 0.1 microg/l from both sets of lysimeters, and thus no significant difference between the two lysimeter sets was shown. However, in both sets of lysimeters several single findings at concentrations above 0.1 microg/l was seen, which might be due to the leaching of particle-bound compounds. A significant difference between the soil residual concencentrations of AMPA was seen, the higher concentration was found in the set of lysimeter where low-tillage had been practiced and where Round Up had been used several times in the years before sampling of the lysimeter soil.     

The response of Lemna trisulca L. to cadmium

Lemna trisulca was grown, using aseptic culture techniques in a filter-sterilized medium, a portion of which was replaced regularly during experiments. L. trisulca responded to the addition of 0.64 microM Cd with a reduction in multiplication rate (MR) 2 +/- 1 days after exposure. The internal Cd content reached 1000 +/- 140 microg Cd/g (dry wt) within 2 days exposure to 0.64 microM Cd. The final yield was reduced by an average of c.8% for each day of exposure to 0.64 microM Cd in a 14 day experiment. This implies that an equilibration period should be used for short-term bioassay tests before the effect of a toxicant is determined. Pretreating L. trisulca with 0.08 or 0.32 microM Cd for 6 weeks had no significant effect on MR or Cd uptake when plants were subsequently exposed to a range of Cd concentrations or grown in a control medium. This suggests that L. trisulca does not become acclimated to elevated Cd concentrations. The MR of L. trisulca fluctuated over a period of almost 600 days and the doubling time ranged from 1.6 to 2.4 days. This produced more than a fivefold difference in final yield in experiments of 14 days duration. The reduction in MR in response to 0.32 microM Cd during this same 600 days period averaged 24% with a coefficient of variation of 38%, and varied with the MR of control cultures. Fluctuations in the intrinsic growth rate and the effect of a toxicant on L. trisulca could potentially confound the assessment of toxicity and must be carefully considered when designing test protocols for aquatic plants.