Sublethal Tests Research Articles

Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae.

The ecological risks posed by two β-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1-1000μgL-1) under standard laboratory conditions for 96h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10μgL-1 of CPX were lower compared to controls (Dunnett post hoc p<0.05) and presented signs of emaciation. Concentrations of 1000μgL-1of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10μgL-1 of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.

Relevant, sensitive, non-standard sublethal tests as aquatic ecotoxicity biomarkers

Relevant, sensitive, non-standard sublethal tests as aquatic ecotoxicity biomarkers

Bioassays with terrestrial and aquatic species as monitoring tools of hydrocarbon degradation.

In this study chemical analyses and ecotoxicity tests were applied for the assessment of a heavily hydrocarbon-contaminated soil prior and after the application of a remediation procedure that consisted in the stimulation of soil autochthonous populations of hydrocarbon degraders in static-ventilated biopiles. Terrestrial bioassays were applied in mixtures of test soils and artificial control soil and studied the survival and reproduction of Eisenia fetida and the avoidance response of E. fetida and Folsomia candida. Effects on aquatic organisms were studied by means of acute tests with Vibrio fischeri, Raphidocelis subcapitata, and Daphnia magna performed on aqueous elutriates from test soils. The bioremediation procedure led to a significant reduction in the concentration of hydrocarbons (from 34264 to 3074mgkg(-1), i.e., 91% decrease) and toxicity although bioassays were not able to report a percentage decrease of toxicity as high as the percentage reduction. Sublethal tests proved the most sensitive terrestrial bioassays and avoidance tests with earthworms and springtails showed potential as monitoring tools of hydrocarbon remediation due to their high sensitivity and short duration. The concentrations of hydrocarbons in water extracts from test soils were 130 and 100μgL(-1) before and after remediation, respectively. Similarly to terrestrial tests, most aquatic bioassays detected a significant reduction in toxicity, which was almost negligible at the end of the treatment. D. magna survival was the most affected by soil elutriates although toxicity to the crustacean was associated to the salinity of the samples rather than to the concentration of hydrocarbons. Ecotoxicity tests with aqueous soil elutriates proved less relevant in the assessment of hydrocarbon-contaminated soils due to the low hydrosolubility of hydrocarbons and the influence of the physicochemical parameters of the aquatic medium.

Uji Toksisitas Letal dan Subletal Logam Berat Merkuri (Hg) Terhadap Ikan Nilem (Osteochilus hasselti)

Mercury (Hg) is a heavy metal could pollute the river. Hg impacts on aquatic organism chronically. The influence of chemical toxic on aquatic organisms could be determined using toxicity (lethal and sublethal) tests. Osteochilus hasselti could be an object for toxicity test. A research, aiming to find LC50-96h concentration, was to find letal and subletal effects of Hg on erythrocyte and hematocryte changes. An experimental method applied Completely Randomized Design. The research was devided into 3 stages, i.e. preliminary, lethal toxicity (LC50-96h) and sublethal toxicity tests, in triplicates. Sampling of sublethal test was performed after an exposure time of 96 h (4 days) and 288 h (12 days). Lethal toxicity test data were analyzed as probit and data from sublethal toxicity test were F-tested. The result showed that LC50-96h of Hg on Osteochilus hasselti was 0.39 mg/L. The sublethal effect of Hg decreased erythrocyte ad hematocryte counts in parallel with increasing its concentration and its exposure time.Keywords: Hg, toxicity, LC50-96 hours, erythrocytes, hematocrits.

Toxic Effects of Emerging Pollutants in Juveniles of the Freshwater GastropodPhysa acuta(Draparnaud, 1805)*

Abstract: In this work, the toxicity of 11 products (three brands of mouthwash, three toothpastes, three detergents and two analgesics) was evaluated in relation to juvenile snails Physa acuta (Draparnaud, 1805). Bioassays of toxicity were conducted and the organisms exposed to six concentrations of each product, along with a control group, to determine the 96-hour LC50 (Lethal Concentration 50). The sensitivity of the gastropods to the different products was determined by comparing their LC50 values to that of acute toxicity values. In addition, the degree of lipoperoxidation in the gastropod tissue was evaluated by measuring the concentration of MDA (malondialdehyde) in sublethal tests (LC10) for chronic toxicity (during 30-day exposure). The toxicity of the products tested varied from highly toxic and harmful (mouthwashes) through harmful (detergents), potentially harmful (analgesics), and non-toxic (toothpastes). The degree of lipoperoxidation observed in the sublethal tests was (from most to least to...

Testing the joint effects hypothesis of elemental defense using Spodoptera exigua.

Metal hyperaccumulation may be an elemental defense, in which high concentrations of a metal in plant tissues decrease herbivore survival or growth rate. The Joint Effects Hypothesis suggests that a combination of metals, or a combination of a metal with an organic compound, may have an enhanced defensive effect. The enhancement may be additive or synergistic: in either case the concentration of a particular metal necessary to provide a defensive benefit for the plant is lowered. We tested the Joint Effects Hypothesis using Spodoptera exigua (beet armyworm) neonates fed artificial diets. Metal + metal experiments utilized diets amended with metal pairs, using four metals commonly hyperaccumulated by plants (Co, Cu, Ni, and Zn). We also conducted metal + organic compound experiments, pairing each metal with nicotine, mustard seed powder, or tannic acid. We tested for joint effects using both lethal (LC20 levels) and sublethal concentrations (10-25% reduced larval weight) of the chemicals tested. For all experiments, either additive or synergistic effects were found. Of the metal + metal pairs tested, three (Co + Cu, Cu + Zn, and Ni + Zn) were synergistic in lethal concentration tests and only Co + Cu was synergistic in sublethal tests. For metal + organic combination lethal tests, synergism occurred for all combinations except for Co or Ni + nicotine, Ni + mustard seed powder, and Zn + nicotine. For sublethal tests, Zn + all three organic chemicals, Co + mustard seed powder or tannic acid, and Cu + nicotine, were synergistic. These results support the Joint Effects Hypothesis, suggesting that metals combined with other metals or organic compounds may be more effective against herbivores than individual metals.

In vivo assessment of DNA damage in Cyprinus carpio after exposure to potassium dichromate using RAPD

The present study used the random amplification of polymorphism DNA (RAPD) technique to evaluate possible DNA damage in the blood cells of Cyprinus carpio exposed to potassium dichromate. Test specimens were exposed to 3 sublethal test concentrations (93.95, 187.9, and 281.85 mg L^{?1}) of potassium dichromate for a period of 96 h. The samplings were collected at 24, 48, 72, and 96 h after exposure. DNA was isolated from the blood samples of both the control and treated groups of C. carpio, and 3 selected decamer primers were used for RAPD-PCR. In comparison to the control, potassium dichromate-treated groups showed differences in RAPD profiles with respect to the disappearance of several bands and the appearance of new ones. There was a significant (P < 0.05) difference between 72 and 96 h after exposure with respect to genomic template stability. The variation in RAPD profiles indicated both time- and concentration-dependent relationships. The results suggested significant genomic template instability as a measurement of DNA damage and further demonstrated the potential of RAPD as a powerful tool for detecting genotoxicity induced by potassium dichromate in aquatic environments.

Physiological effects of paraquat in juvenile African catfish Clarias gariepinus (Burchel 1822)

Objective: To investigate the physiological effects of paraquat in African freshwater catfish Clarias gariepinus. Methods: Two sublethal test concentrations of paraquat (1.37 and 2.75 mg/L) were chosen based on the 96 h LC50 value (27.46 mg/L). Some experimental fish were exposed to these concentrations and control group for 15 d. Peripheral blood samplings were taken at intervals for assessment of haematological and biochemical parameters. Results: Exposure to paraquat affected behaviour and morphology of Clarias gariepinus. There were significant decreases (P<0.05) in the mean values of hemoglobin, red blood cells, packed cell volume, cellular hemoglobin, and cellular hemoglobin concentration. The levels of white blood cells, glucose, aspartate aminotransferase, and alanine aminotransferase significantly increased (P<0.05) while protein levels declined. However, no definite pattern of changes was observed in the number and type of leucocytes. Conclusions: The results of the present study indicate that paraquat is toxic and has the potential to impair on the physiological activities in African catfish Clarias gariepinus. The use of paraquat should be strongly controlled and carefully monitored to avoid the possible damage done to the environment.

A highly efficient nonchemical method for isolating live nematodes (Caenorhabditis elegans) from soil during toxicity assays.

The success of soil toxicity tests using Caenorhabditis elegans may depend in large part on recovering the organisms from the soil. However, it can be difficult to learn the International Organization for Standardization/ASTM International recovery process that uses the colloidal silica flotation method. The present study determined that a soil-agar isolation method provides a highly efficient and less technically demanding alternative to the colloidal silica flotation method. Test soil containing C. elegans was arranged on an agar plate in a donut shape, a linear shape, or a C curve; and microbial food was placed outside the soil to encourage the nematodes to leave the soil. The effects of ventilation and the presence of food on nematode recovery were tested to determine the optimal conditions for recovery. A linear arrangement of soil on an agar plate that was sprinkled with microbial food produced nearly 83% and 90% recovery of live nematodes over a 3-h and a 24-h period, respectively, without subjecting the nematodes to chemical stress. The method was tested using copper (II) chloride dihydrate, and the resulting recovery rate was comparable to that obtained using colloidal silica flotation. The soil-agar isolation method portrayed in the present study enables live nematodes to be isolated with minimal additional physicochemical stress, making it a valuable option for use in subsequent sublethal tests where live nematodes are required.

Sublethal Effects of Methanolic Extract of Raphia hookeri on the Reproductive Capacity of Clarias gariepinus

Raphia hookeri fruits are used for fishing in Nigeria due to their ichthyotoxic properties. This study investigated the toxic effects of R. hookeri on the reproductive capacity of Clarias gariepinus. The results from both short-term (96-hour test) and long-term (3-month sublethal test) bioassays revealed a linear relationship between R. hookeri extract dose and negative effects on the catfish. The percentage survival of both sexes of the catfish decreased with increasing extract concentration at short-term exposure, with LC50 values of 600 mg/L and 800 mg/L for male and female, respectively. At long-term exposure, the reproductive capacity of 10–12-month-old male and female brood-stocks diminished at relatively higher concentrations of R. hookeri fruit extract, with the gravid females producing fewer and mostly unviable eggs. The fruit extract also affected the eggs’ hatchability and fry survival when the exposed gravid females were treated with pituitary hormone and sperms from unexposed males, while the exposed males were unable to sexually stimulate female brooders. Sperms and pituitary hormone from exposed males were infertile, leading to low percentage of hatched eggs and mortality of the few hatched fries within 24 hours. These results confirmed the ethnobotanical use of this fruit extract for fishing in Nigeria.

Assessment of fetal exposure risk following seminal excretion of a therapeutic IgG4 (T-IgG4) monoclonal antibody using a rabbit model

The Folsomia candida reproduction test [ISO, 1998. Soil quality—Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants. International Standard Organization Report 11267, 1998, Geneva] is used to evaluate the ecotoxicological risks of contaminants in soils. The aim of this study was to compare the sensitivity of growth and reproduction of F. candida to four xenobiotics: two metals (Cd, Al), one metalloid (As), and one organic compound (pentachlorophenol). We showed that reproduction is a slightly more sensitive parameter than growth: EC20 for reproduction was 1.25 μg/g dry soil for arsenic, 56 μg/g for cadmium, 97.5 μg/g for aluminum, and 41.7 μg/g for pentachlorophenol. The corresponding EC20 values for growth were 2.8, 65, 630, and 94.6 μg/g. Keeping in mind that a growth test needs fewer juveniles and less time than a reproduction test, we conclude that the two parameters are complementary and could be used for a better ecotoxicological evaluation of contaminants. However, the relative growth and reproduction sensitivities should be tested with more chemicals before growth could be considered as a good alternative for a faster sublethal test.

Assessment of genotoxic and mutagenic potential of hexavalent chromium in the freshwater fish Labeo rohita (Hamilton, 1822)

The present study was undertaken to investigate the genotoxicity and mutagenicity of sublethal concentrations of hexavalent chromium (potassium dichromate) in the Indian major carp, Labeo rohita. The 96 h LC50 value of potassium dichromate estimated was 118 mg L−1 by probit analysis using SPSS (version 16.0) software. Based on 96 h LC50 value, three sublethal test concentrations of potassium dichromate (29.5, 59.0 and 88.5 mg L−1) were selected and specimens were exposed in vivo to these test concentrations for 96 h. The mutagenic and genotoxic effects of potassium dichromate were evaluated in gill and blood cells using micronucleus (MN) test and comet assay. In general, significant (p < 0.05) effects due to the concentrations and the exposure durations were observed in exposed specimens. The MN induction was highest at 96 h at all the test concentrations in the peripheral blood. A similar trend was observed for the DNA damage, measured in terms of percentage of tail DNA, in erythrocyte and gill cells. The study indicated hazardous effect of the hexavalent chromium to fish and other aquatic organisms and indirectly to human beings.

Induction of micronuclei and nuclear lesions in Channa punctatus following exposure to carbosulfan, glyphosate and atrazine

The genotoxic effects of commonly used agricultural pesticides viz., carbosulfan, glyphosate, and atrazine, were evaluated in Channa punctatus (Pisces, Perciformes) using micronucleus (MN) test and induction of nuclear lesions (NL). The 96 h LC50 value were estimated by probit analysis as 0.27, 32.0 and 42.0 mg L−1, respectively, for carbosulfan, glyphosate, and atrazine using semi-static bioassays. Based on these values, three sublethal test concentrations of carbosulfan (0.07, 0.13, 0.20 mg L−1), glyphosate (8.1, 16.3, 24.4 mg L−1) and atrazine (10.6, 21.2, 31.8 mg L−1) corresponding to ¼, ½ and ¾ of the LC50 of the pesticides respectively, were selected for exposure for 96 h. Peripheral blood samplings were taken at intervals of 24 h for assessment of MN and NL frequencies. Considerably higher genotoxic damage was induced by carbosulfan as compared to glyphosate and atrazine. There were significant effects (p < 0.01) of concentrations in all the treated groups. The induction of MN and NL was highest at 96 h pesticide exposure at all test concentrations. The nuclear abnormalities recorded in this study, such as blebbed-, lobed-, notched- and bi-nuclei, other than micronuclei, are indicators of genotoxic damage.

Lethal and behavioral effects of pesticides on the insect predator Macrolophus pygmaeus

Macrolophus pygmaeus (Hemiptera: Miridae) is a common generalist predator in Mediterranean agro-ecosystems. We evaluated the lethal effects of six insecticides and a fungicide on M. pygmaeus nymphs exposed to the pesticides through three routes of exposure: direct, residual and oral. Chlorantraniliprole and emamectin-benzoate caused less than 25% mortality to M. pygmaeus and were classified as harmless according to the International Organization for Biological Control rating scheme. In contrast, thiacloprid and metaflumizone caused 100% and 80% mortality, respectively, and were classified as harmful. Indoxacarb and spinosad resulted in close to 30% mortality to the predator, and were classified as slightly harmful, while the fungicide copper hydroxide caused 58% mortality and was rated as moderately harmful. Chlorantraniliprole and thiacloprid were selected for further sublethal testing by exposing M. pygmaeus to two routes of pesticide intake: pesticide residues and feeding on sprayed food. Thiacloprid led to an increase in resting and preening time of the predator, and a decrease in plant feeding. Chlorantraniliprole resulted in a decrease in plant feeding, but no other behaviors were affected. In addition, thiacloprid significantly reduced the predation rate of M. pygmaeus, whereas chlorantraniliprole had no significant effect on predation rate. The results of the study suggest that thiacloprid is not compatible with M. pygmaeus, while further research needs to be carried out for metaflumizone and copper hydroxide. All other products seem to be relatively compatible with M. pygmaeus, though studies on their sublethal effects will shed more light into their safety.

Characterization of carboxylesterase in skin mucus of Cirrhinus mrigala and its assessment as biomarker of organophosphate exposure

Presence of carboxylesterase (CbE) activity in the skin mucus of Cirrhinus mrigala was investigated. CbE activity in skin mucus showed higher substrate preference for α-naphthyl acetate over p-nitrophenyl acetate. Four CbE isozymes-CbE-1, CbE-2, CbE-3, and CbE-4 were observed in skin mucus during zymography. The isozyme CbE-4 was characterized as typical serine esterase, whereas CbE-1, CbE-2, and CbE-3 were identified as sulphhydryl group-dependent serine esterases. In vitro treatment of skin mucus with the organophosphorus insecticide, Nuvan(®) showed strong inhibition of CbE activity. In vivo exposure of the fish to sublethal test concentrations (5 and 15mg/l) of the insecticide also revealed significant inhibition of CbE activity in mucus. After the cessation of exposure, CbE activity recovered to its control level during the recovery periods. Thus, CbE activity in skin mucus could be considered a biomarker of the organophosphorus insecticide exposure to fish and a useful tool in monitoring environmental toxicity.

Derivation of South African water quality guidelines for Roundup® using species sensitivity distribution

Glyphosate-based herbicides are among the leading products used in South Africa to control weeds and invading alien plant species. Although these herbicides ultimately find their way into aquatic ecosystems, South Africa has no water quality guideline based on indigenous species to protect the country's aquatic biota against these biocides. In this study, South African water quality guidelines (SAWQGs) for Roundup® based on species sensitivity distribution (SSD) using indigenous aquatic biota were developed. Short-term and long-term toxicity tests were conducted with eight different aquatic species belonging to five different taxonomic groups. Static non-renewal experimental methods were employed for short-term lethal tests (≤4 days), and static renewal for long-term sublethal tests (≥4 days ≤21 days). LC50 values for animal exposure and EC50 values for algae were calculated using probit analysis and linear regression of transformed herbicide concentration as natural logarithm data against percentage growth inhibition, respectively. No effect concentration (NEC) was determined based on the dynamic energy budget model, using survival data. The LC50, EC50 and NEC values were used to develop species sensitivity distribution (SSD) concentrations for Roundup®. Based on the SSD concentrations, the short-term and long-term SAWQGs for Roundup® were derived as 0.250 (0.106–0.589) mg/L, and 0.002 (0.000–0.021) mg/L, respectively. These WQGs may be useful in protecting South African aquatic life against transient or long-term exposure to glyphosate-based chemicals as part of integrated water resources management.

DNA damage and oxidative stress modulatory effects of glyphosate-based herbicide in freshwater fish, Channa punctatus

The present study was undertaken to evaluate the genotoxic and oxidative stress modulatory effects of commercial formulation of glyphosate-based herbicide (Roundup®) in freshwater fish Channa punctatus. Three sublethal test concentrations of the herbicide viz., SL-I (1/10th of LC50=∼3.25mgL−1), SL-II (1/8th of LC50=∼4.07mgL−1) and SL-III (1/5th of LC50=∼6.51mgL−1) were calculated using 96-LC50 value and the test specimens were exposed to these concentrations. Blood and gill cells of the exposed specimens were sampled on day 1, 7, 14, 21, 28 and 35 to examine the DNA damage using comet assay and to assess the alteration in lipid peroxidation and antioxidant enzymes activities. The highest DNA damage was observed on day 14 at all test concentrations followed by gradual non-linear decline. Induction of oxidative stress in the blood and gill cells were evidenced by increased lipid peroxidation level, while antioxidants namely superoxide dismutase, catalase and glutathione reductase responded in a concentration-dependent manner. The results supported the integrated use of comet and antioxidant assays in determining the toxicity of water pollutants which could be used as part of monitoring programs.

Toxicity of the ionophore antibiotic lasalocid to soil-dwelling invertebrates: Avoidance tests in comparison to classic sublethal tests

Lasalocid is a veterinary ionophore antibiotic used for prevention and treatment of coccidiosis in poultry. It enters the environment with the use of contaminated manure on agricultural land. Despite its extensive use, the effects of lasalocid on non-target soil organisms are poorly explored. We used classical subleathal ecotoxicity tests to assess the effects of lasalocid on earthworms (Eisenia andrei) and isopods (Porcellio scaber) and compared the results with tests using avoidance behaviour as the endpoint. The results showed that avoidance is a much more sensitive endpoint. For earthworms, EC50 for avoidance (12.3mgkg−1 dry soil) was more than five times lower than EC50 for reproduction (69.6mgkg−1 dry soil). In isopods the sensitivity of the behavioural response test was even higher. While the highest lasalocid concentration 202mgkg−1 had no significant effects on isopod growth or survival, already the lowest used concentration in the behavioural assay (4.51mgkg−1) caused significant impact on isopod behaviour. Using the avoidance test results for calculating the predicted no-effect concentration (PNEC) of lasalocid to soil invertebrates, the value is close to the predicted environmental concentration (PEC). This indicates that the use of lasalocid-contaminated manure could potentially impair the habitat function of agricultural soils.

Genotoxicity and antioxidant enzyme activity induced by hexavalent chromium in Cyprinus carpio after in vivo exposure

Fish, being an important native of the aquatic ecosystem, are exposed to multipollution states and are therefore considered as model organisms for ecotoxicological studies of aquatic pollutants, including metal toxicity. We investigated oxidative stress (OS) in liver, kidney and gill tissues through antioxidant enzyme activities and genotoxicity induced in whole blood and gill tissues through comet assay and micronucleus (MN) test in Cyprinus carpio after 96-hour in vivo static exposure to potassium dichromate at three sublethal (SL) test concentrations, including SL-I [93.95 mg/L, i.e. one quarter of half-maximal lethal concentration (LC50)], SL-II (187.9 mg/L, i.e. one half of LC50), and SL-III (281.85 mg/L, i.e. three quarters of LC50), along with a control. The 96-hour LC50 value for potassium dichromate was estimated to be 375.8 mg/L in a static system in the test species. Tissues samples were collected at 24, 48, 72 and 96 hours postexposure. Results indicated that the exposed fish experienced OS as characterized by significant (p < 0.05) variation in antioxidant enzyme activities, as compared to the control. Activities of superoxide dismutase and glutathione peroxidase increased, whereas activity of catalase decreased with the progression of the experiment. The mean percent DNA damage in comet tail and MN induction in gills and whole blood showed a concentration-dependent increase up to 96-hour exposure. The findings of this study would be helpful in organ-specific risk assessment of Cr(VI)-induced OS and genotoxicity in fishes.

Toxic Effects of Acetochlor on Mortality, Reproduction and Growth of Caenorhabditis elegans and Pristionchus pacificus

The effects of acetochlor on the mortality, growth and reproduction of two nematode species were assessed. The LC50 values for Caenorhabditis elegans and Pristionchus pacificus were 1,296 and 210.7mg/L at 24h, and 540.0 and 126.4mg/L at 48h exposure, respectively. In three succession generations, reproductive capacity was more sensitive in P. pacificus than in C. elegans. Moreover, the sublethal test endpoint of final length was more sensitive with P. pacificus. This study suggested that acetochlor had no long-term effects on C. elegans at lower concentrations. The higher concentrations of acetochlor (from 40 to 160mg/L) revealed sublethal toxicity to the two tested species, with P. pacificus being more sensitive than C. elegans.