Irrigation runoff insecticide pollution of rivers in the Imperial Valley, California (USA)

The Alamo and New Rivers located in the Imperial Valley, California receive large volumes of irrigation runoff and discharge into the ecologically sensitive Salton Sea. Between 1993 and 2002 we conducted a series of studies to assess water quality using three aquatic species: a cladoceran (Ceriodaphnia dubia), a mysid (Neomysis mercedis), and a larval fish (Pimephales promelas). Although no mortality was observed with the P. promelas, high-level toxicity to the invertebrate species was documented in samples from both rivers during many months of each year. Toxicity identifications and chemical analyses identified the organophosphorus insecticides (OP), chlorpyrifos and diazinon, as the cause of C. dubia toxicity. The extent of the C. dubia mortality was highly correlated with quantities of these OPs applied in the river watersheds. C. dubia mortality occurred during more months of our 2001/2002 study than in the 1990s investigations. During 2001/2002, the extensive C. dubia mortality observed in New River samples was caused by OP insecticide pollution that originated from Mexico. Mortality to N. mercedis in New River samples was likely caused by contaminants other than OP insecticides. Our studies document OP insecticide-caused pollution of the Alamo River over a 10-year period and provide the necessary information for remediation efforts.     

Response of Ceriodaphnia dubia to ionic silver: discrepancies among model predictions, measured concentrations and mortality

Silver thiosulfate, often a waste product of photoprocessing, is less bioavailable or toxic to aquatic organisms than is ionic silver. We conducted duplicate 48-h Ceriodaphnia dubia tests in reconstituted laboratory water using treatments of 92.7 nM Ag+ with various concentrations of thiosulfate. Expected Ag+ concentrations were generated for thiosulfate treatment levels using MINEQL + chemical equilibrium modeling. Ag+ concentrations in treatments were determined using a novel silicon-based sensor. Based on predicted Ag+ and published 48-h LC50 values for C. dubia, we did not expect to observe adverse effects. Yet, 100% mortality was observed at low thiosulfate treatments, whereas > 85% and > 95% survival was observed at higher thiosulfate treatment levels, respectively. Our results indicate that biotic responses match the sensor-based Ag+ concentrations. However, there is a discrepancy between these empirical results and responses expected to occur with Ag+ concentrations as predicted by MINEQL + chemical modeling. By correlating silicon sensor data with toxicity results obtained from our laboratory, our work clearly relates a specific chemical form (Ag+) to toxicity results.     

Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors

Glyphosate-based herbicides (e.g. Roundup) are extensively used in the aquatic environment, but there is a paucity of data on the toxicity of the formulated products and the influences by environmental factors. In this study, the acute toxicity of technical-grade glyphosate acid, isopropylamine (IPA) salt of glyphosate, Roundup and its surfactant polyoxyethylene amine (POEA) to Microtox bacterium (Vibrio fischeri), microalgae (Selenastrum capricornutum and Skeletonema costatum), protozoa (Tetrahymena pyriformis and Euplotes vannus) and crustaceans (Ceriodaphnia dubia and Acartia tonsa) was examined and the relative toxicity contributions of POEA to Roundup were calculated. The effects of four environmental factors (temperature, pH, suspended sediment and algal food concentrations) on the acute toxicity of Roundup to C. dubia were also examined. Generally, the toxicity order of the chemicals was: POEA>Roundup>glyphosate acid>IPA salt of glyphosate, while the toxicity of glyphosate acid was mainly due to its high acidity. Microtox bacterium and protozoa had similar sensitivities towards Roundup toxicity (i.e. IC50 from 23.5 to 29.5 mg AE/l). In contrast, microalgae and crustaceans were 4-5 folds more sensitive to Roundup toxicity than bacteria and protozoa. Except photosynthetic microalgae, POEA accounted for more than 86% of Roundup toxicity and the toxicity contribution of POEA was shown to be species-dependent. Increase in pH (6-9) and increase of suspended sediment concentration (0-200 mg/l) significantly increased the toxicity of Roundup to C. dubia, but there were no significant effects due to temperature change and food addition.     

Integrated assessment of the impacts of agricultural drainwater in the Salinas River (California,USA)

The Salinas River is the largest of the three rivers that drain into the Monterey Bay National Marine Sanctuary in central California. Large areas of this watershed are cultivated year-round in row crops and previous laboratory studies have demonstrated that acute toxicity of agricultural drainwater to Ceriodaphnia dubia is caused by the organophosphate (OP) pesticides chlorpyrifos and diazinon. In the current study, we used a combination of ecotoxicologic tools to investigate incidence of chemical contamination and toxicity in waters and sediments in the river downstream of a previously uncharacterized agricultural drainage creek system. Water column toxicity was investigated using a cladoceran C. dubia while sediment toxicity was investigated using an amphipod Hyalella azteca. Ecological impacts of drainwater were investigated using bioassessments of macroinvertebrate community structure. The results indicated that Salinas River water downstream of the agricultural drain is acutely toxic to Ceriodaphnia, and toxicity to this species was highly correlated with combined toxic units (TUs) of chlorpyrifos and diazinon. Laboratory tests were used to demonstrate that sediments in this system were acutely toxic to H. azteca, which is a resident genus. Macroinvertebrate community structure was moderately impacted downstream of the agricultural drain input. While the lowest macroinvertebrate abundances were measured at the station demonstrating the greatest water column and sediment toxicity and the highest concentrations of pesticides, macroinvertebrate metrics were more significantly correlated with bank vegetation cover than any other variable. Results of this study suggest that pesticide pollution is the likely cause of laboratory-measured toxicity in the Salinas River samples and that this factor may interact with other factors to impact the macroinvertebrate community in the system.     

Feasibility of constructed wetlands for removing chlorothalonil and chlorpyrifos from aqueous mixtures

Chlorpyrifos (an insecticide) and chlorothalonil (a fungicide) are transported in stormwater runoff and can be lethal to receiving aquatic system biota. This study determined removal rates of chlorpyrifos and chlorothalonil in simulated stormwater runoff treated in constructed wetland mesocosms. Using sentinel species, Ceriodaphnia dubia and Pimephales promelas, observed declines in toxicity of the simulated runoff after treatment were 98 and 100%, respectively. First order removal rates were 0.039/h for chlorpyrifos and 0.295/h for chlorothalonil in these experiments. Constructed wetland mesocosms were effective for decreasing concentrations of chlorpyrifos and chlorothalonil in simulated stormwater runoff, and decreasing P. promelas and C. dubia mortality resulting from these exposures. The results from this study indicate that constructed wetlands could be part of an efficient mitigation strategy for stormwater runoff containing these pesticides.     

Toxicity of prednisolone, dexamethasone and their photochemical derivatives on aquatic organisms

Light exposure of aqueous suspensions of prednisolone and dexamethasone causes their partial phototransformation. The photoproducts, isolated by chromatographic techniques, have been identified by spectroscopic means. Prednisolone, dexamethasone and their photoproducts have been tested to evaluate their acute and chronic toxic effects on some freshwater chain organisms. The rotifer Brachionus calyciflorus and the crustaceans Thamnocephalus platyurus and Daphnia magna were chosen to perform acute toxicity tests, while the alga Pseudokircheneriella subcapitata (formerly known as Selenastrum capricornutum) and the crustacean Ceriodaphnia dubia to perform chronic tests. The photochemical derivatives are more toxic than the parent compounds. Generally low acute toxicity was found. Chronic exposure to this class of pharmaceuticals caused inhibition of growth population on the freshwater crustacean C. dubia while the alga P. subcapitata seems to be less affected by the presence of these drugs.     

Waterborne and sediment toxicity of fluoxetine to select organisms

Ecological risk assessments of pharmaceuticals are currently difficult because little-to-no aquatic hazard and exposure information exists in the peer-reviewed literature for most therapeutics. Recently several studies have identified fluoxetine, a widely prescribed antidepressant, in municipal effluents. To evaluate the potential aquatic toxicity of fluoxetine, single species laboratory toxicity tests were performed to assess hazard to aquatic biota. Average LC(50) values for Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas were 0.756 (234 microg/l), 2.65 (820 microg/l), and 2.28 microM (705 microg/l), respectively. Pseudokirchneriella subcapitata growth and C. dubia fecundity were decreased by 0.044 (14 microg/l) and 0.72 microM (223 microg/l) fluoxetine treatments, respectively. Oryias latipes survival was not affected by fluoxteine exposure up to a concentration of 28.9 microM (8.9 mg/l). An LC(50) of 15.2 mg/kg was estimated for Chironomus tentans. Hyalella azteca survival was not affected up to 43 mg/kg fluoxetine sediment exposure. Growth lowest observed effect concentrations for C. tentans and H. azteca were 1.3 and 5.6 mg/kg, respectively. Our findings indicate that lowest measured fluoxetine effect levels are an order of magnitude higher than highest reported municipal effluent concentrations.     

Reduction in labile copper in the 7-day Ceriodaphnia dubia toxicity test due to the interaction with zooplankton food

Due to the increased popularity of zooplankton toxicity tests, it is important to investigate potential confounding factors. Though zooplankton food has been studied extensively to meet the nutritional needs of the zooplankton, less research has been done on whether food addition reduces the toxicity of metals in the tested sample. This investigation combines toxicity tests and metal speciation analysis to determine whether the EPA recommended food of YCT (yeast, cerophyll, and trout chow) and Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) reduces copper toxicity by decreasing the concentration of labile copper. Toxicity tests were performed with Ceriodaphnia dubia on culture water spiked with 0, 787, and 1574 nM copper with five different food levels. A Chelex-100 cation exchange resin and a graphite furnace-atomic absorption spectrophotometer were used in conjunction with the toxicity tests to measure the amount of labile copper in the culture water. At the EPA recommended food dosage, the C. dubia food has a chelating capacity of approximately 500 nM Cu. For both concentrations of spiked culture water, the toxicity to C. dubia was reduced with increasing food level, which seemed to be both from a decrease in labile copper concentration and an increase in the nutritional condition of the zooplankton.     

锐颈特与马拉硫磷交替使用对麦穗鱼脑乙酰胆碱酯酶活性恢复的 …

研究了５％锐颈特县浮剂与９５％马拉硫磷文替使用对麦穗鱼（Ｐｓｅｕｄｏｒａｓｂｏｒａ ｐａｒｏａ）乙酰胆碱酯酶（ＡＣｈＥ,ＥＣ３．１．１．７）活性的影响。结果表明,０．２ｍｇ／Ｌ,锐颈物与２ｍｇ／Ｌ马拉硫磷交替使用均影响被抑制的ＡＣｈＥ活性恢复。文中还探讨了研究农药影响对于合理评价农药环境毒性的意义。     

In situ exposures using caged organisms: a multi-compartment approach to detect aquatic toxicity and bioaccumulation

An in situ toxicity and bioaccumulation assessment approach is described to assess stressor exposure and effects in surface waters (low and high flow), the sediment-water interface, surficial sediments and pore waters (including groundwater upwellings). This approach can be used for exposing species, representing major functional and taxonomic groups. Pimephales promelas, Daphnia magna, Ceriodaphnia dubia, Hyalella azteca, Hyalella sp., Chironomus tentans, Lumbriculus variegatus, Hydra attenuatta, Hexagenia sp. and Baetis tibialis were successfully used to measure effects on survival, growth, feeding, and/or uptake. Stressors identified included chemical toxicants, suspended solids, photo-induced toxicity, indigenous predators, and flow. Responses varied between laboratory and in situ exposures in many cases and were attributed to differing exposure dynamics and sample-processing artifacts. These in situ exposure approaches provide unique assessment information that is complementary to traditional laboratory-based toxicity and bioaccumulation testing and reduce the uncertainties of extrapolating from the laboratory to field responses.     

Effects of prochloraz and malathion on the red-legged partridge: a semi-natural field study

A semi-natural field study was carried out to assess the likelihood of a potentiation of toxicity between the ergosterol biosynthesis inhibiting (EBI) fungicide, prochloraz, and the organophosphorus (OP) insecticide, malathion, in the red-legged partridge (Alectoris rufa). Groups of partridges kept in four large grassland enclosures were exposed to either prochloraz-treated or control wheat for 7 days after which two of the enclosures were sprayed with malathion whilst the remaining two were sham-sprayed. Cytochrome P-450, aldrin epoxidase and 7-ethoxyresorufin-O-deethylase (EROD) activities were found to be significantly higher in the group exposed to prochloraz alone compared to controls, suggesting that induction of the hepatic microsomal monooxygenase system had occurred by ingestion of prochloraz-treated wheat. However, the level of induction produced was not sufficient to cause a potentiation of malathion toxicity. There was evidence for induction of several forms of P-450 recognised by antibodies raised against 1A1, 2C6 and 4A1 in the prochloraz-exposed partridges.     

Does malaoxon play a role in the geno- and cytotoxic effects of malathion on human choriocarcinoma cells?

This investigation was undertaken to elucidate whether the active metabolite of malathion, malaoxon, has any role in exerting cyto- and genotoxic effects for human choriocarcinoma (JAR) cell line which is an acceptable model for human placental cells. Gas chromatography-mass spectrometry (GC-MS) analysis were separately performed on the cell compartment and supernatant cell culture medium after subjecting the cell line to different malathion concentrations (10-400 μg/mL) and for various incubation periods (0.5 to 24 hours). GC-MS analysis showed that the sonication performed for the disruption of the cells did not cause the chemical change of malathion. The uptake of malathion by the cells was relatively fast. However, the presence of malaoxon, even in trace amounts, could not be confirmed either in samples originating from disrupted cells or in the cell culture medium. Although the hydrolysis of malaoxon occurred in the culture medium, this degradation process could not be counted as a reason for the absence of malaoxon. Since both malathion and malaoxon standard compounds could be accurately detected and distinguished by the applied liquid-liquid extraction and GC-MS methods, one can conclude that, in the case of JAR cells, the parent compound, (i.e. malathion itself) is responsible for the observed in vitro cyto- and genotoxic effects. Our results indicate that the direct toxicity of malathion contributes to the complications of pregnancy observed for environmental malathion exposure.     

Protective effect of Syzygium cumini against pesticide-induced cardiotoxicity

Pesticide-induced toxicity is a serious issue which has resulted in plethora of diseases all over the world. The organophosphate pesticide malathion has caused many incidents of poisoning such as cardiac manifestations. The present study was designed to evaluate the effect of Syzygium cumini on malathion-induced cardiotoxicity. Dose optimization of malathion and polyphenols such as curcumin, (?)-epicatechin, gallic acid, butylated hydroxyl toluene, etc. was done by MTT cell proliferation assay. Nuclear deformities, ROS production, and integrity of extra cellular matrix components were analyzed by different techniques. S. cumini methanolic pulp extract (MPE), a naturally derived gallic acid-enriched antioxidant was taken to study its effect on malathion-induced toxicity. Nuclear deformities, ROS production, and integrity of extra cellular matrix components were also analyzed. Twenty micrograms per milliliter LD50 dose of malathion was found to cause stress-mediated responses in H9C2 cell line. Among all the polyphenols, gallic acid showed the most significant protection against stress. Gallic acid-enriched methanolic S. cumini pulp extract (MPE) showed 59.76 %?±?0.05, 81.61 %?±?1.37, 73.33 %?±?1.33, 77.19 %?±?2.38 and 64.19 %?±?1.43 maximum inhibition for DPPH, ABTS, NO, H₂O₂ and superoxide ion, respectively, as compared to ethanolic pulp extract and aqueous pulp extract. Our study suggests that S. cumini MPE has the ability to protect against the malathion-mediated oxidative stress in cardiac myocytes.     

Surface metal adsorption on zooplankton carapaces: implications for exposure and effects in consumer organisms

The current study aimed to determine the potential of two important aquatic invertebrate crustacean species, Daphnia magna and Ceriodaphnia dubia, to adsorb cadmium on to their carapaces from aqueous solution. Using the Langmuir equation to model data outputs, it was shown that cadmium readily became associated with the carapace surfaces of both species, with uptake being dependent on exposure time and concentration. Maximum carapace-adsorption potential was found to be directly related to surface area, so that at predicted carapace saturation, D. magna neonates bound approximately five times more cadmium than the smaller C. dubia neonates. However, adsorption per unit surface area was found to be similar under the same exposure conditions. Results of surface metal adsorption studies in C. dubia suggested that short term exposures to high concentrations of aqueous cadmium would lead to similar levels of adsorption as obtained with long-term exposures to low concentrations. The study illustrates that contaminants adsorbed to prey surfaces may be an important mechanism of exposure to predators, and highlights some potential problems of feeding organisms during long-term toxicity tests.     

Does malaoxon play a role in the geno- and cytotoxic effects of malathion on human choriocarcinoma cells?

This investigation was undertaken to elucidate whether the active metabolite of malathion, malaoxon, has any role in exerting cyto- and genotoxic effects for human choriocarcinoma (JAR) cell line which is an acceptable model for human placental cells. Gas chromatography-mass spectrometry (GC-MS) analysis were separately performed on the cell compartment and supernatant cell culture medium after subjecting the cell line to different malathion concentrations (10–400 μg/mL) and for various incubation periods (0.5 to 24 hours). GC-MS analysis showed that the sonication performed for the disruption of the cells did not cause the chemical change of malathion. The uptake of malathion by the cells was relatively fast. However, the presence of malaoxon, even in trace amounts, could not be confirmed either in samples originating from disrupted cells or in the cell culture medium. Although the hydrolysis of malaoxon occurred in the culture medium, this degradation process could not be counted as a reason for the absence of malaoxon. Since both malathion and malaoxon standard compounds could be accurately detected and distinguished by the applied liquid-liquid extraction and GC-MS methods, one can conclude that, in the case of JAR cells, the parent compound, (i.e. malathion itself) is responsible for the observed in vitro cyto- and genotoxic effects. Our results indicate that the direct toxicity of malathion contributes to the complications of pregnancy observed for environmental malathion exposure.     

Influence of glyphosate and its formulation (Roundup) on the toxicity and bioavailability of metals to Ceriodaphnia dubia

This study examined the toxicological interaction between glyphosate (or its formulation, Roundup) and several heavy metals to a freshwater cladoceran, Ceriodaphnia dubia. We demonstrated that all binary combinations of Roundup and metals (Cd, Cu, Cr, Ni, Pb, Se and Zn) exhibited "less than additive" mixture toxicity, with 48-h LC50 toxic unit > 1. Addition of glyphosate alone could significantly reduce the acute toxicity of Ag, Cd, Cr, Cu, Ni, Pb and Zn (but not Hg and Se). The ratio between glyphosate and metal ions was important in determining the mitigation of metal toxicity by glyphosate. A bioaccumulation study showed that in the presence of glyphosate the uptake of some metals (e.g. Ag) was halted but that of others (e.g. Hg) was increased significantly. Therefore, our study strongly suggests that glyphosate and its commercial formulations can control the toxicity as well as the bioavailability of heavy metals in aquatic ecosystems where both groups of chemicals can co-occur.     

Growth,photosynthesis and removal responses of the cyanobacteria Chroococcus sp. to malathion and malaoxon

Abstract

Malathion is an organophosphorus pesticide widely used in agricultural crops, despite its toxicity. In addition, malaoxon occurs by oxidation of malathion being more toxic. The toxic effects of malathion and malaoxon in humans include hepatoxicity, breast cancer, genetic damage and endocrine disruption. The aim of this study involved assessing the effect of malathion commercial grade on Chroococcus sp., and its potential as an alternative to the removal of this pesticide and its transformation product such as malaoxon. We evaluated the effect of malathion at different concentrations (1, 25, 50, 75 and 100?ppm) on the biomass of the cyanobacteria Chroococcus sp. grown in medium BG-11; also, we analyse its ability to degrade both malathion and malaoxon into a temperature of 28?±?2?°C and at pH 6. The results showed that 50?ppm of malathion the cyanobacteria Chroococcus sp. reached the highest removal efficiency of malathion and malaoxon (69 and 65%, respectively); also, the growth rate of Chroococcus sp. increased without inhibiting the production of chlorophyll “a”, this can be explained by the hormesis phenomenon. Therefore, we consider that the cyanobacteria Chroococcus sp. may be a good candidate for bioremediation of aquatic systems contaminated with organophosphorus pesticides such as malathion and its transformation product such as malaoxon.     

Tolerance and acclimation to zinc of Ceriodaphnia dubia

Zinc is an essential metal for all living organisms. However, so far, little or no attention has been paid to the consequences of zinc deficiency or acclimation to this metal during culturing and testing on toxicity test results. In this study, the cladoceran Ceriodaphnia dubia was acclimated for 10 generations to four zinc concentrations ranging from 0 to 100 microg Zn/l and changes in zinc tolerance were monitored using acute (48 h) and chronic (9 days) assays. C. dubia deprived of zinc and acclimated to 13 microg Zn/l had a lower fitness in comparison with organisms acclimated to 50 and 100 microg Zn/l. In the two lowest versus the two highest acclimation concentrations the 9dEC50 values (on immobility) were 358-387 microg Zn/l versus 486-489 microg Zn/l; the mean number of young per female was 11-18 versus 25-32; and the time to first brood was 4.7-5.0 days versus 4.0-4.3 days. Moreover, the coefficient of variation of all parameters tested was highest in the two lowest acclimation concentrations. The results indicate that culturing test animals in media lacking trace metals such as zinc could give rise to animals that are unnaturally sensitive to those same metals daring toxicity tests.     

Effects of malathion and chlorpyrifos on acetylcholinesterase and antioxidant defense system in Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

The study was undertaken to evaluate the effects of malathion and chlorpyrifos on acetylcholinesterase (AChE), esterase (EST) activity and antioxidant system after topical application with different concentration to Oxya chinensis. The results showed that malathion and chlorpyrifos inhibited EST, AChE activity and increased malondialdehyde (MDA) contents. A change in superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR) activity combined with reduced glutathione (GSH) and total glutathione (tGSH) contents was found in O. chinensis after malathion and chlorpyrifos treatments. Malathion and chlorpyrifos increased SOD and CAT activity compared with the control. With the concentrations increasing, SOD and CAT activity showed the similar tendency, namely, SOD and CAT activity increased at the lower concentrations and decreased at the higher concentrations. The results showed that malathion and chlorpyrifos decreased significantly GR activity. GST and GPx activity at the studied concentrations of chlorpyrifos was lower than that of the control. However, no significance was observed. GPx and GST activity in malathion treated grasshoppers showed a biphasic response with an initial increase followed by a decline in its activity. Malathion and chlorpyrifos decreased GSH contents and the ratio of GSH/GSSG. The present findings indicated that the toxicity of malathion and chlorpyrifos might be associated with oxidative stress.     

Photodegradation of organophosphorus insecticides - investigations of products and their toxicity using gas chromatography-mass spectrometry and AChE-thermal lens spectrometric bioassay

Four organophosphorus compounds: azinphos-methyl, chlorpyrifos, malathion and malaoxon in aqueous solution were degraded by using a 125 W xenon parabolic lamp. Gas chromatography-mass spectrometry (GC-MS) was used to monitor the disappearance of starting compounds and formation of degradation products as a function of time. AChE-thermal lens spectrometric bioassay was employed to assess the toxicity of photoproducts. The photodegradation kinetics can be described by a first-order degradation curve C=C0e(-kt), resulting in the following half lives: 2.5min for azinphos-methyl, 11.6 min for malathion, 13.3 min for chlorpyrifos and 45.5 min for malaoxon, under given experimental conditions. During the photoprocess several intermediates were identified by GC-MS suggesting the pathway of OP degradation. The oxidation of chlorpyrifos results in the formation of chlorpyrifos-oxon as the main identified photoproduct. In case of malathion and azinphos-methyl the corresponding oxon analogues were not detected. The formation of diethyl (dimethoxy-phosphoryl) succinate in traces was observed during photodegradation of malaoxon and malathion. Several other photoproducts including trimethyl phosphate esters, which are known to be AChE inhibitors and 1,2,3-benzotriazin-4(3H)-one as a member of triazine compounds were identified in photodegraded samples of malathion, malaoxon, and azinphos-methyl. Based on this, two main degradation pathways can be proposed, both result of the (P-S-C) bond cleavage taking place at the side of leaving group. The enhanced inhibition of AChE observed with the TLS bioassay during the initial 30 min of photodegradation in case of all four OPs, confirmed the formation of toxic intermediates. With the continuation of irradiation, the AChE inhibition decreased, indicating that the formed toxic compounds were further degraded to AChE non-inhibiting products. The presented results demonstrate the importance of toxicity monitoring during the degradation of OPs in processes of waste water remediation, before releasing it into the environment.