The use of a lacertid lizard as a model for reptile ecotoxicology studies: part 2--biomarkers of exposure and toxicity among pesticide exposed lizards

As part of a wider study examining the impacts of corn pesticides on lacertid lizards in north-western Portugal, we examined various physiological, biochemical, and histological biomarkers of exposure and effect among field populations of Podarcis bocagei. Biomarkers included body condition index, standard metabolic rate, locomotor performance, parasitization, glutathione oxidative pathways and related enzyme activity, lipid peroxidation and liver and testis histology. Few of the various biomarkers investigated provided statistically significant evidence of toxic effect. However, using a weight of evidence approach, we conclude that pesticides are affecting lizards living in the vicinity of pesticide exposed corn agriculture sites. Lizards from these locations present a profile of animals under metabolic stress with reduced condition indices, increased standard metabolic rate, lower incidence of hepatocyte vacuolation, altered iron metabolism, increased activation of GSH oxidation pathways, and even increased prevalence of hemoparasites.     

Using organic-certified rather than synthetic pesticides may not be safer for biological control agents: selectivity and side effects of 14 pesticides on the predator Orius laevigatus

The generalist predator Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) is a key natural enemy of various arthropods in agricultural and natural ecosystems. Releases of this predator are frequently carried out, and it is included in the Integrated Pest Management (IPM) programs of several crops. The accurate assessment of the compatibility of various pesticides with predator activity is key for the success of this strategy. We assessed acute and sublethal toxicity of 14 pesticides on O. laevigatus adults under laboratory conditions. Pesticides commonly used in either conventional or organic farming were selected for the study, including six biopesticides, three synthetic insecticides, two sulfur compounds and three adjuvants. To assess the pesticides’ residual persistence, the predator was exposed for 3 d to pesticide residues on tomato sprouts that had been treated 1 h, 7 d or 14 d prior to the assay. The percentage of mortality and the sublethal effects on predator reproductive capacity were summarized in a reduction coefficient (E_x) and the pesticides were classified according to the IOBC (International Organization for Biological Control) toxicity categories. The results showed that the pesticides greatly differed in their toxicity, both in terms of lethal and sub lethal effects, as well as in their persistence. In particular, abamectin was the most noxious and persistent, and was classified as harmful up to 14 d after the treatment, causing almost 100% mortality. Spinosad, emamectin, metaflumizone were moderately harmful until 7 d after the treatment, while the other pesticides were slightly harmful or harmless. The results, based on the combination of assessment of acute mortality, predator reproductive capacity pesticides residual and pesticides residual persistence, stress the need of using complementary bioassays (e.g. assessment of lethal and sublethal effects) to carefully select the pesticides to be used in IPM programs and appropriately time the pesticides application (as function of natural enemies present in crops) and potential releases of natural enemies like O. laevigatus.     

Inhibition of plasma butyrylcholinesterase activity in the lizard Gallotia galloti palmae by pesticides: a field study

A field study was performed to evaluate the effect of exposure to organophosphorus (OP) and carbamate (CB) pesticides on the lizard Gallotia galloti palmae. Butyrylcholinesterase (BChE) activity was measured in the plasma of 420 lizards collected from agricultural and reference areas on the Island of La Palma (Canary Islands, Spain) in two sampling periods. Exposure to cholinesterase-inhibiting pesticides was evaluated by a statistical criterion based on a threshold value (two standard deviations below the mean enzyme activity) calculated for the reference group, and a chemical criterion based on the in vitro reactivation of BChE activity using pyridine-2-aldoxime methochloride (2-PAM) or after water dilution of the sample. Mean (+/- SD) BChE activity for lizards from agricultural areas was significantly lower (Fuencaliente site = 2.00 +/- 0.98 micromol min(-1) ml(-1), Tazacorte site = 2.88 +/- 1.08) than that for lizards from the reference areas (Los Llanos site = 3.06 +/- 1.17 micromol min(-1) ml(-1), Tigalate site = 3.96 +/- 1.62). According to the statistical criterion, the number of lizards with BChE depressed was higher at Fuencaliente (22% of males and 25.4% of females) than that sampled at Tazacorte (7.8% of males and 6.2% of females). According to the chemical criterion, Fuencaliente also yielded a higher number of individuals (112 males and 47 females) with BChE activity inhibited by both OP and CB pesticides. CBs appeared to be the pesticides most responsible for BChE inhibition because most of the samples showed reactivation of BChE activity after water treatment (63.3% from Fuencaliente and 29% from Tazacorte). We concluded that the use of reactivation techniques on plasma BChE activity is a better and more accurate method for assessing field exposure to OP/CB pesticides in this lizard species than making direct comparisons of enzyme activity levels between sampling areas.     

Survival and behavior of the insecticide-exposed predators Podisus nigrispinus and Supputius cincticeps (Heteroptera: Pentatomidae)

Pentatomid stinkbugs are important predators of defoliating caterpillars in agricultural and forestry systems, and knowledge of the impact of insecticides on natural enemies is important information for integrated pest management (IPM) programs. Thus, we assessed the toxicity and behavioral sublethal response of the predators Podisus nigrispinus and Supputius cincticeps exposed to deltamethrin, methamidophos, spinosad and chlorantraniliprole, insecticides commonly used to control the velvetbean caterpillar (Anticarsia gemmatalis) in soybean crops. With the exception of deltamethrin for S. cincticeps, all insecticides showed higher acute toxicity to the prey than to these natural enemies providing effective control of A. gemmatalis. The recommended field concentration of deltamethrin, methamidophos and spinosad for controlling A. gemmatalis caused 100% mortality of P. nigrispinus and S. cincticeps nymphs. Chlorantraniliprole was the less toxic and the most selective insecticide to these predators resulting in mortalities of less than 10% when exposed to 10× the recommended field concentration for a period of 72 h. Behavioral pattern changes in predators were found for all insecticides, especially methamidophos and spinosad, which exhibited irritability (i.e., avoidance after contact) to both predator species. However, insecticide repellence (i.e., avoidance without contact) was not observed in any of the insects tested. The lethal and sublethal effects of pesticides on natural enemies is of great importance for IPM, and our results indicate that substitution of pyrethroid and organophosphate insecticides at their field rates by chlorantraniliprole may be a key factor for the success of IPM programs of A. gemmatalis in soybeans.     

Leaf litter leachates have the potential to increase lifespan, body size, and offspring numbers in a clone of Moina macrocopa

Leaf litter processing is one major pathway of the global organic carbon cycle. During this process, a variety of small reactive organic compounds are released and transported to the aquatic environment, and may directly impact aquatic organisms as natural xenobiotics. We hypothesize that different forest stockings produce different leachate qualities, which in turn, stress the aquatic communities and, eventually, separate sensitive from tolerant species. Particularly, leachates from coniferous trees are suspected to have strongly adverse impacts on sensitive species. We exposed individuals of a clone of the model organism, Moina macrocopa, to comparable concentrations (approximately 2 mM) of litter leachates of Norway spruce, Picea abies, Colorado blue spruce, Picea pungens, black poplar, Populus nigra, and sessile oak, Quercus petraea. The animals were fed ad libitum. The following life trait variables were recorded: growth, lifespan, and lifetime offspring. To identify, whether or not exposure to litter leachates provokes an internal oxidative stress in the exposed animals we measured the superoxide anion radical scavenging capacity via photoluminescence. Except of P. abies, exposure to the leachates reduced this antioxidant capacity by approximately 50%. Leachate exposures, except that of Quercus, increased body size and extended lifespan; furthermore, particularly the leachates of both Picea species significantly increased the offspring numbers. This unexpected behavior of exposed Moina may be based on food supplements (e.g., high carbohydrate contents) in the leachates or on yet to be identified regulatory pathways of energy allocation. Overall, our results suggest that the potentially adverse effects of litter leachates can be overruled by either bacterial-growth supporting fractions in the leachates or an internal compensation mechanism in the Moina individuals.     

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.     

Comparing the sensitivity of soil invertebrates to pesticides with that of Eisenia fetida

The sole routine testing of the standard earthworm Eisenia fetida for the terrestrial risk assessment of pesticides has been under much debate since other soil invertebrates may be more sensitive than this standard test species. However, the very low availability of laboratory toxicity data for taxa other than E. fetida has greatly hampered sensitivity comparisons. In the present study, the relative tolerance (T_rel) approach was used to enable comparing toxicity thresholds obtained from the US-EPA ECOTOX database, for main terrestrial taxonomic groups and pesticidal types of action (insecticides, fungicides, herbicides, and other) separately. Analyses confirmed previously reported lower and higher sensitivity of collembolans to fungicides and insecticides, respectively. However, various other discrepancies in susceptibility relative to E. fetida were encountered as indicated by species sensitivity distributions and/or calculated 95% confidence intervals of T_rel values. Arachnids and isopods were found to be more sensitive to insecticides, and nematodes to fungicides, as compared to E. fetida. Implications of study findings for the terrestrial risk assessment of pesticides are discussed.     

A comparison of mixture toxicity assessment: examining the chronic toxicity of atrazine, permethrin and chlorothalonil in mixtures to Ceriodaphnia cf. dubia

Pesticides predominantly occur in aquatic ecosystems as mixtures of varying complexity, yet relatively few studies have examined the toxicity of pesticide mixtures. Atrazine, chlorothalonil and permethrin are widely used pesticides that have different modes of action. This study examined the chronic toxicities (7-d reproductive impairment) of these pesticides in binary and ternary mixtures to the freshwater cladoceran Ceriodaphnia cf. dubia. The toxicity of the mixtures was compared to that predicted by the independent action (IA) model for mixtures, as this is the most appropriate model for chemicals with different modes of action. Following this they were compared to the toxicity predicted by the concentration addition (CA) model for mixtures. According to the IA model, the toxicity of the chlorothalonil plus atrazine mixture conformed to antagonism, while that of chlorothalonil and permethrin conformed to synergism. The toxicity of the atrazine and permethrin mixture as well as the ternary mixture conformed to IA implying there was either no interaction between the components of these mixtures and/or in the case of the ternary mixture the interactions cancelled each other out to result in IA. The synergistic and antagonistic mixtures deviated from IA by factors greater than 3 and less than 2.5, respectively. When the toxicity of the mixtures was compared to the predictions of the CA model, the binary mixture of chlorothalonil plus atrazine, permethrin plus atrazine and the ternary mixture all conformed to antagonism, while the binary mixture of chlorothalonil plus permethrin conformed to CA. Using the CA model provided estimates of mixture toxicity that did not markedly underestimate the measured toxicity, unlike the IA model, and therefore the CA model is the most suitable to use in ecological risk assessments of these pesticides.     

Histopathological effects of carbaryl on testes of snake-eyed lizard, Ophisops elegans

Background, aim and scope  

Due to their ecological niche and insectivore nature, lizards are of increased risk of exposure to pesticides in agricultural areas. In addition to their potential direct effects on non-target species, insecticides can also result in indirect impacts on lizard population by reducing their food source. Carbaryl is a common insecticide that is widely used in areas of Turkey that are home to a variety of reptiles. However, to date, little is known about the potential effects of the exposure of reptiles such as lizards or snakes to this pesticide. The aim of the study was to investigate toxic effects of carbaryl on the testes of snake-eyed lizard, Ophisops elegans that is common to regions in Turkey where Carbaryl is applied and that it can be easily cultured in the laboratory.     

Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris

Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg⁻¹ chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (≤1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field.     

Lethal effects of abamectin on the aquatic organisms Daphnia similis, Chironomus xanthus and Danio rerio

Abamectin is used as an acaricide and insecticide for fruits, vegetables and ornamental plants, as well as a parasiticide for animals. One of the major problems of applying pesticides to crops is the likelihood of contaminating aquatic ecosystems by drift or runoff. Therefore, toxicity tests in the laboratory are important tools to predict the effects of chemical substances in aquatic ecosystems. The aim of this study was to assess the potential hazards of abamectin to the freshwater biota and consequently the possible losses of ecological services in contaminated water bodies. For this purpose, we identified the toxicity of abamectin on daphnids, insects and fish. Abamectin was highly toxic, with an EC₅₀ 48 h for Daphnia similis of 5.1 ng L⁻¹, LC₅₀ 96 h for Chironomus xanthus of 2.67 μg L⁻¹ and LC₅₀ 48 h for Danio rerio of 33 μg L⁻¹.     

Absorption of current use pesticides by snapping turtle (Chelydra serpentina) eggs in treated soil

Reptiles often breed within agricultural and urban environments that receive frequent pesticide use. Consequently, their eggs and thus developing embryos may be exposed to pesticides. Our objectives were to determine (i) if turtle eggs are capable of absorbing pesticides from treated soil, and (ii) if pesticide absorption rates can be predicted by their chemical and physical properties. Snapping turtle (Chelydra serpentina) eggs were incubated in soil that was treated with 10 pesticides (atrazine, simazine, metolachlor, azinphos-methyl, dimethoate, chlorpyrifos, carbaryl, endosulfan (I and II), captan, and chlorothalonil). There were two treatments, consisting of pesticides applied at application rate equivalents of 1.92 or 19.2 kg a.i/ha. Eggs were removed after one and eight days of exposure and analyzed for pesticides using gas chromatography coupled with a mass selective detector (GC-MSD) or high performance liquid chromatography (HPLC). Absorption of pesticides in eggs from soil increased with both magnitude and duration of exposure. Of the 10 pesticides, atrazine and metolachlor generally had the greatest absorption, while azinphos-methyl had the lowest. Chlorothalonil was below detection limits at both exposure rates. Our preliminary model suggests that pesticides having the highest absorption into eggs tended to have both low sorption to organic carbon or lipids, and high water solubility. For pesticides with high water solubility, high vapor pressure may also increase absorption. As our model is preliminary, confirmatory studies are needed to elucidate pesticide absorption in turtle eggs and the potential risk they may pose to embryonic development.     

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate “sink” for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 μg L⁻¹) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO₃. Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.     

Rapid biotransformation of arsenic by a model protozoan Tetrahymena pyriformis GL-C. [corrected

Arsenic biomethylation and biovolatilization are thought to be two important metabolic pathways in aquatic and soil environments. Tetrahymena thermophila is a genus of free-living ciliated protozoan that is widely distributed in freshwater environments around the world. In this study, we studied arsenic accumulation, speciation, efflux, methylation and volatilization in this unicellular eukaryote exposed to various concentrations of arsenate. Our results show that T. thermophila accumulated 187 mg.kg⁻¹ dry weight of arsenic when exposed to 40 μM for 48 h, with MMAs(V) (monomethylarsenate) and DMAs(V) (dimethylarsenate) as the dominant species, accounting for 66% of the total arsenic. Meanwhile, arsenate, arsenite, MMAs(V) and DMAs(V) were detected in the culture medium; the last three were released by the cells. The production of volatile arsenic increased with increasing external As(V) concentrations and exposure time. To our knowledge, this is the first study on arsenic metabolism, particularly biomethylation and biovolatilization, in protozoa.     

Salicylic acid involved in the regulation of nutrient elements uptake and oxidative stress in Vallisneria natans (Lour.) Hara under Pb stress

In this study, the alterations in nutrient elements content, reactive oxygen species level and antioxidant response were studied in leaves of Vallisneria natans (Lour.) Hara exposed to salicylic acid (SA, 10 or 100 μM), or Pb (50 μM) or their combinations for 4 d. No significant alterations in Mn and Ca content were observed but content of Cu, Zn, Fe and P decreased in plants exposed to SA alone. SA application inhibited the uptake of Pb and partially reversed Pb-induced the alterations in Mn, Ca and Fe content in leaves of V. natans exposed to 50 μM Pb. The decreased chlorophyll (a + b) and increased malondialdehyde and O²⁻ and H₂O₂ content were detected in plants exposed to 100 μM SA, 50 μM Pb, 10 μM SA + 50 μM Pb or 100 μM SA + 50 μM Pb. Application SA partially inhibited Pb-induced the increase of malondialdehyde, O²⁻ and H₂O₂ content. 100 μM SA decreased the activity of NADH oxidase and the content of non-protein thiols, carotenoids and ascorbic acid and increased the content of dehydroascorbate in plants treated with or without Pb. SA alone decreased the ascorbate peroxidase activity and increased the catalase and peroxidase activity, while SA application increased catalase activity but had no significant effect on ascorbate peroxidase and peroxidase activity in V. natans exposed to Pb. The results indicate that SA involves in the regulation of Pb uptake, nutrient balance and oxidative stress.     

A list of fish species that are potentially exposed to pesticides in edge-of-field water bodies in the European Union—a first step towards identifying vulnerable representatives for risk assessment

Surrogate species are used in standard toxicity tests for the environmental risk assessment of chemicals. Test results are then extrapolated to the situation in the field, which is often associated with a large degree of uncertainty. Since a vulnerable species in the field is not only characterised by its intrinsic sensitivity to a stressor but also by its potential for exposure and its population resilience, the identification of focal species based on these three components of vulnerability is needed for a more ecologically relevant risk assessment. This study listed European fish species that are susceptible to pesticide exposure in the field and thus achieved the first step towards identifying focal species for the risk assessment of pesticides for fish in Europe. A step-wise filtering approach was applied to list freshwater fish species that are native to Europe and widespread in the European Union, which inhabit streams, ditches or ponds in agricultural landscapes and therefore, are at an elevated risk of being exposed to pesticides. Out of the 579 fish species occurring in European freshwater, 27 species met the filtering criteria. The resulting list was verified based on monitoring studies that were conducted in agricultural landscapes over the past 20 years. Focal fish species that can be used for a more ecologically relevant environmental risk assessment of pesticides in Europe can be identified from the produced list of species by further assessing their ecological (life history and dispersal characteristics) and intrinsic sensitivities.     

Sampling atmospheric pesticides with SPME: Laboratory developments and field study

To estimate the atmospheric exposure of the greenhouse workers to pesticides, solid phase microextraction (SPME) was used under non-equilibrium conditions. Using Fick's law of diffusion, the concentrations of pesticides in the greenhouse can be calculated using pre-determined sampling rates (SRs). Thus the sampling rates (SRs) of two modes of SPME in the lab and in the field were determined and compared. The SRs for six pesticides in the lab were 20.4-48.3 mL min⁻¹ for the exposed fiber and 0.166-0.929 mL min⁻¹ for the retracted fiber. In field sampling, two pesticides, dichlorvos and cyprodinil were detected with exposed SPME. SR with exposed SPME for dichlorvos in the field (32.4 mL min^-1) was consistent with that in the lab (34.5 mL min^-1). SR for dichlorvos in the field (32.4 mL min⁻¹) was consistent with that in the lab (34.5 mL min⁻¹). The trends of temporal concentration and the inhalation exposure were also obtained.     

The effect of insecticides on the non-target predatory mite Kampimodromus aberrans: Laboratory studies

The side-effects of pesticides on predatory mites have been investigated at various levels and international teams (e.g., the IOBC/wprs Working Group “Pesticides and beneficial organisms”) have selected a few species of predatory mites occurring in Central and Northern Europe as representative for such studies. Key biocontrol species occurring in Southern Europe have received much less attention. Kampimodromus aberrans is the most important predator of herbivorous mites in South-European vineyards treated with selective pesticides. The impact of pesticides on K. aberrans populations has been studied in field conditions whereas few toxicological tests have been conducted in the laboratory because of difficulties in rearing this species. A method for rearing K. aberrans in the laboratory has recently been set up allowing toxicological studies to be conducted. In this paper, a toxicological method to assess the effects of pesticides on K. aberrans is described and the effects of insecticides frequently used in European vineyards on two K. aberrans strains are reported. These strains were collected from vineyards treated with organophosphates. Insecticides characterized by different modes of action were selected for trials. Among these, etofenprox and spinosad were classified as harmful to predatory mites. Chlorpyrifos reduced predatory mite fecundity, and was classified as moderately harmful for both strains. The toxicity of thiamethoxam and flufenoxuron varied with the strain (low to moderate). Indoxacarb and methoxyfenozide appeared to be harmless or slightly harmful. Implications of this study for adopting IPM tactics with a reduced risk for K. aberrans are discussed.     

Environmental and lifestyle factors for organochlorine exposure among women living in Southern Spain

Southern Spain has the largest area of intensive greenhouse agriculture in Europe, and may constitute a special case of occupational and female exposure, because this type of farming requires considerable pesticide use and employs many women. We measured adipose tissue levels of 14 organochlorine pesticides in 458 women living in this area and analyzed the relationship between pesticide level/presence and sociodemographic characteristics, reproductive history or life-style factors that may influence this exposure. Pesticide presence was determined by gas chromatography with electron-capture detector. All fat samples were positive for 1 residue. DDT or metabolites were found in 98.25%, with mean value of 660 ng g⁻¹ of lipid. p,p′-DDE level was higher in women who were older, with lower educational level or obese. Almost 70% had measurable levels of endosulfan and/or metabolites, with a mean total value of 38.8 ng g⁻¹ of lipid. Endosulfan-I exposure was higher in women with shorter residence in rural settings and more frequent in women with 3 children. 52.62% were exposed to 1 of aldrin–dieldrin–endrin group. Endrin was more frequent in women who were younger, with higher educational level or no agricultural working experience; dieldrin was more frequent in women who were older, with lower educational level or more children. Finally, lindane residues were found in 39.30%. Lindane levels were higher in women who breastfed longer or had more children. Research is required on women occupationally exposed to a selected group of organochlorine pesticides, especially those of reproductive age, as a basis for preventive action.     

Changes in Lumbriculus variegatus metabolites under hypoxic exposure to benzo(a)pyrene,chlorpyrifos and pentachlorophenol: Consequences on biotransformation

The regulation of endogenous metabolites is still not fully understood in aquatic invertebrates exposed concurrently to toxicants and hypoxia. Despite the prevalence of hypoxia in the aquatic environment, toxicity estimations seldom account for multiple stressors thereby differing from natural conditions. In this study, we examined the influence of hypoxia (<30% O₂) on contaminant uptake and the composition of intracellular metabolites in Lumbriculus variegatus exposed to benzo(a)pyrene (B(a)P, 3 μg L⁻¹), chlorpyrifos (CPF, 100 μg L⁻¹) or pentachlorophenol (PCP, 100 μg L⁻¹). Tissue extracts of worms were analyzed for 123 metabolites by gas chromatography–mass spectrometry and metabolite levels were then related to treatments and exposure time. Hypoxia markedly increased the accumulation of B(a)P and CPF, which underlines the significance of oxygen in chemical uptake. The oxygen effect on PCP uptake was less pronounced. Succinate and glycerol-3-phosphate increased significantly (p < 0.0001) following hypoxic treatment, whereas sugars, cysteine, and cholesterol were effectively repressed. The buildup of succinate coupled with the corresponding decline in intracellular 2-oxo- and 2-hydroxy glutaric acid is indicative of an active hypoxia inducible factor mechanism. Glutamate, and TCA cycle intermediates (fumarate, and malate) were disturbed and evident in their marked suppression in worms exposed concurrently to hypoxia and PCP. Clearly, hypoxia was the dominant stressor for individuals exposed to B(a)P or CPF, but to a lesser extent upon PCP treatment. And since oxygen deprivation promotes the accumulation of different toxicants, there may be consequences on species composition of metabolites in natural conditions.