Multigenerational exposure of Ag materials (nano and salt) in soil - environmental hazards in Enchytraeus crypticus (Oligochaeta).

Because of its properties, silver is among the most used metals both as salt and as nanomaterials (NMs), hence reaching the environment. Multigenerational (MG) exposure testing is scarce, and especially so for NMs and soil invertebrates. In this study the MG effects of Ag NMs (Ag NM300K) and Ag salt (AgNO3) were assessed, using Enchytraeus crypticus in LUFA 2.2 soil. Survival, reproduction and internal Ag concentration in the animals were measured throughout 7 generations (5 generations (F0-F4) in spiked soil plus 2 (F5-F6) in clean soil) exposed to sublethal concentrations corresponding to the reproduction EC10 and EC50 obtained in standard toxicity tests (45 and 60 mg Ag per kg soil DW for AgNO3; 20 and 60 mg Ag per kg soil DW for Ag NM300K). MG exposure caused a dose-related decrease in reproduction for both Ag forms. Ag uptake peaked in the F1 (64 days) for AgNO3 and F2 (96 days) for Ag NM300K, after which it decreased. In agreement with toxicokinetic studies, a maximum body Ag concentration was reached (20 mg Ag per kg body DW (AgNO3) and 70 mg Ag per kg body DW (Ag NM300K)) and after which detoxification mechanisms seem to be activated with elimination of Ag accompanied by a decrease in reproduction. Transfer to clean soil allowed Ag to be (fully) eliminated from the animals. This MG study confirmed the effects determined in standard reproduction toxicity tests but further allowed to monitor the dynamics between exposure and effects of the Ag materials, and how the animals seem to cope with Ag for 7 generations by compensating between detoxification and reproductive output.

Toxicokinetics and toxicodynamics of Ag nanomaterials (NM300K) in the soil environment-impact on Enchytraeus crypticus (Oligochaeta).

Silver (Ag) is one of the most used elements in the nanomaterials (NMs) form, which upon release to the environment can be harmful to organisms. We compared the toxicokinetics (TK) and toxicodynamics (TD) of Ag from AgNO3 (0, 15, 45, 135, 405 mg Ag/kg soil) and AgNM300K (0, 75, 150, 300, 600, 1200 mg Ag/kg soil) in the model organism Enchytraeus crypticus. Organisms were exposed in LUFA 2.2 soil, and besides body Ag concentrations, survival and reproduction were determined, in a time series (for 21 days). In the soil, the available (CaCl2 extractable) Ag fraction from Ag NM300K increased from 0 to 21 days but did not consistently change for AgNO3. Internal concentrations reached equilibrium in most exposures to both Ag forms. The organisms were able to internalize and eliminate Ag, but less when exposed to Ag NM300K. The overall uptake rate constants for Ag from AgNO3 and Ag NM300K exposures were 0.05 and 0.06 kg soil/kg organism/day, respectively, the elimination rate constants 0.2 and 0.1 day-1, respectively. For AgNO3 the median lethal concentrations decreased steadily with time, while for Ag NM300K they remained constant during the first 10 days of exposure followed by a 2-fold decline in the last 7 days. The 21-d LC50s for both Ag forms were similar but the LC50inter (based on internal concentrations) were 63 and 121 mg Ag/kg body DW (Dry Weight) for AgNO3 and Ag NM300K, respectively, showing higher toxicity of AgNO3. These results show the importance of assessing time to toxicity, a relevant factor in toxicity assessment, especially for NMs.

Toxicokinetics and toxicodynamics of chromium in the soil invertebrate Enchytraeus crypticus (Oligochaeta).

Chromium emissions led to increased concentrations in soil, where it can affect soil organisms to relevant levels. With the aim of better understanding the effects of Cr throughout time, its toxicokinetics-toxicodynamics (TKTD) were evaluated in the soil model organism Enchytraeus crypticus to assess the development of internal concentrations and consequent toxic effects. To achieve this goal, organisms were exposed in LUFA 2.2 soil spiked with increasing CrCl3 concentrations. During the 21-day exposure period, survival, internal concentrations, and reproduction were evaluated at several time points up to 21 days. Uptake and elimination rate constants were 0.0044 kg soil/kg organism/day and 0.023 per day, respectively. Internal Cr concentrations increased with time, generally reaching equilibrium within 14 days with an estimated LC50inter (based on internal metal concentrations) of 57.7 mg Cr/kg body DW. Internal Cr concentrations were regulated by the organisms up to exposure to 360 mg Cr/kg soil DW, where the elimination rate was highest, but at 546 mg Cr/kg soil DW the animals were no longer able to eliminate Cr, and the internal concentrations were well above the estimated LC50inter. At day 21, exposure to 546 mg Cr/kg soil DW significantly reduced survival by 23 %, while reproduction EC50 was 344 mg Cr/kg soil DW. This study highlights the advantages of using a TKTD approach to understand the development of internal metal concentrations in time and relate it to the phenotypical effects observed. Toxicity is better understood when also taking into account time and not just exposure concentration alone.

Toxicokinetics and toxicodynamics of copper and cadmium in the soil invertebrate Enchytraeus crypticus (Oligochaeta).

The aim of this study was to evaluate the toxicokinetics-toxicodynamics (TKTD) of Cu and Cd in the soil model organism Enchytraeus crypticus, and assess the development of internal effect concentrations over time. Animals were exposed in LUFA 2.2 soil spiked with increasing concentrations of Cu and Cd. Survival, reproduction and internal metal concentrations in the animals were evaluated at different points in time over a period of 21 days. Internal concentrations increased with time, for Cu reaching a steady state after c. 10 days, except for the highest test concentration, and for Cd continuing to increase after 21 days. Applying a one-compartment model to all data together, estimated uptake and elimination rate constants for Cu and Cd were 0.08 and 0.45 kg soil/kg organism/day and 0.4 and 0.04 per day, respectively. Median lethal concentrations, based on total soil concentrations, decreased with time for Cu and did not reach a steady state level, but they did not change with time for Cd. The LC50inter (based on internal concentrations) was 75 mg Cu/kg body DW and > 800 mg Cd/kg body weight. Animals were able to regulate Cu internal concentrations, keeping them low, while for Cd internal concentrations continued to increase showing lack of regulation and also the importance of exposure time. This study highlights the advantages of using a TKTD approach to understand the relation between organism survival and internal Cu or Cd concentrations over time.

Toxicokinetics of Chromium in Enchytraeus crypticus (Oligochaeta).

Chromium is naturally occurring, but emission from anthropogenic sources can lead to increased soil concentrations. Information on its toxicokinetics is essential in order to understand the time needed to reach toxicity and the mechanisms of uptake/elimination. In this study the toxicokinetics of Cr(III) was evaluated using the soil standard species Enchytraeus crypticus. The animals were exposed to 180 mg Cr/kg dry soil, a sublethal concentration, in LUFA 2.2 natural soil. OECD guideline 317 was followed, with a 14-day uptake phase in spiked soil followed by a 14-day elimination in clean soil. Exposure to Cr led to fast uptake and elimination, with Ku = 0.012 kgsoil/kgorganism/day and Ke = 0.57 day-1. The bioaccumulation factor was 0.022, and DT50 for elimination was 1.2 days. The concentration of Cr reached an internal equilibrium in the animals after 10 days. Transfer to clean soil allowed body Cr concentrations to return to background levels after approximately 7 days. E. crypticus seemed able to efficiently regulate internal Cr concentrations by actively eliminating Cr (an essential element). Although Ku and Ke deviated from the values reported in other studies for other soil invertebrates, the bioaccumulation factors were similar. These findings show the importance of toxicokinetic studies in evaluating toxicity based on internal metal concentrations that can more accurately represent the bioavailable concentration.

Toxicokinetics of copper and cadmium in the soil model Enchytraeus crypticus (Oligochaeta).

Toxicokinetics information is key to understanding the underlying intoxication processes, although this is often lacking. Hence, in the present study the toxicokinetics of copper (Cu) and cadmium (Cd) was assessed in the soil invertebrate Enchytraeus crypticus. The animals were exposed in LUFA 2.2 natural soil spiked to the estimated EC20 for reproduction effects in the Enchytraeid Reproduction Test (ERT), i.e. 80 mg Cu/kg soil Dry Weight (DW) and 20 mg Cd/kg soil DW. Tests followed the OECD guideline 317, including a 14-day uptake phase in spiked soil followed by 14 days elimination in clean soil, with samplings at days 0, 1, 2, 4, 7, 10, and 14. Exposure to Cu showed fast uptake, reaching a steady state after approx. 7 days, whereas for Cd, internal concentration increased and did not reach a clear steady state even after 14 days. When transferred to clean soil, Cu was rapidly eliminated returning to initial levels, while Cd-exposed animals still contained increased residue levels after 14 days. These differences in toxicokinetics have consequences for the toxicity and toxicodynamics and are indicative of the way essential and non-essential elements are handled by enchytraeids, likely also other soil invertebrates. This argues for the relevancy of longer exposure testing for elements like Cd compared to Cu, where phenotypical effects can well occur later at non-tested periods, e.g. after the 21 days' duration of the standard ERT using E. crypticus.

Multigenerational effects of copper nanomaterials (CuONMs) are different of those of CuCl2: exposure in the soil invertebrate Enchytraeus crypticus.

Nanomaterials (NMs) are recommended to be tested in longer term exposures. Multigenerational (MG) studies are scarce and particularly important because effects can be transferred to the next generation. The current risk assessment framework does not include MG effects and this is a caveat for persistent materials. Here, the effects of copper NMs (CuONMs) and copper salt (CuCl2) were assessed in a MG exposure (4 generations in spiked soil + 2 generations in clean soil, F1 to F7 generations in total), with the standard soil model Enchytraeus crypticus, using relevant reproduction test effect concentrations (EC10, EC50), monitoring survival and reproduction. This represented ca. 1 year continuous exposure tests. MG effects varied with effect concentration and test materials: CuONMs caused increased toxicity for EC10 exposed organisms (EC50 did not change), and transfer to clean media reset effects, whereas CuCl2 reduced toxicity for EC10 and EC50, but the transfer to clean media "revived" the initial effects, i.e. close to EC50 levels in F7. Clearly CuONMs and CuCl2 cause different mechanisms of toxicity or response in the long term, not predictable based on short term or one generation studies. The present contributes for the improvement of risk assessment, adding important information for the long term exposure and effects.

Hazard assessment of nickel nanoparticles in soil-The use of a full life cycle test with Enchytraeus crypticus.

Nanoparticles (NPs) such as nickel (Ni) are widely used in several applications. Nevertheless, the environmental effects of Ni NPs are still poorly understood. In the present study, the toxicity of Ni NPs and nickel nitrate (NiNO3 ) was assessed using the standard test species in soil ecotoxicology, Enchytraeus crypticus (Oligochaeta), in a full life cycle test, adding the endpoints hatching, growth, and time to reach maturity, besides survival and reproduction as in the standard Organisation for Economic Co-operation and Development Guideline 220 and/or International Organization for Standardization 16387. For Ni NPs, the Ni in soil and in soil solution was concentration- and time-dependent, with a relatively higher soil solution content in the lower and shorter exposure concentrations and times. Overall, NiNO3 was more toxic than Ni NPs, and toxicity seemed to occur via different mechanisms. The former caused reduced hatching (50% effect concentration [EC50] = 39 mg Ni/kg soil), and the negative effects remained throughout the life cycle, in all measured endpoints (growth, maturation, survival, and reproduction). For Ni NPs, hatching was the most sensitive endpoint (EC50 = 870 mg Ni/kg soil), although the organisms recovered; that is, additional endpoints across the life cycle showed that this effect corresponded to a delay in hatching because organisms survived and reproduced at concentrations up to 1800 mg Ni/kg soil. On the other hand, the lowest tested concentration of Ni NPs (100 mg Ni/kg soil) caused reproduction effects similar to those at higher concentrations (1000 and 1800 mg Ni/kg soil). The present results show that the potential implications of a nonmonotonic dose response should be considered when assessing the risks of Ni NP exposure in soil. Environ Toxicol Chem 2017;36:2934-2941. © 2017 SETAC.

Effects of copper oxide nanomaterials (CuONMs) are life stage dependent - full life cycle in Enchytraeus crypticus.

Copper oxide nanomaterials (CuONMs) have various applications in industry and enter the terrestrial environment, e.g. via sewage sludge. The effects of CuONMs and copper chloride (CuCl2) were studied comparing the standard enchytraeid reproduction test (ERT) and the full life cycle test (FLCt) with Enchytraeus crypticus. CuONMs mainly affected growth or juveniles' development, whereas CuCl2 mainly affected embryo development and/or hatching success and adults survival. Compared to the ERT, the FLCt allowed discrimination of effects between life stages and provided indication of the underlying mechanisms; further, the FLCt showed increased sensitivity, e.g. reproductive effects for CuONMs: EC10 = 8 mg Cu/kg and EC10 = 421 mg Cu/kg for the FLCt and the ERT respectively. The performance of the FLCt is preferred to the ERT and we recommend it as a good alternative to assess hazard of NMs. Effects of CuONMs and CuCl2 are life stage dependent and are different between Cu forms.

Enchytraeid Reproduction Test(PLUS): hatching, growth and full life cycle test--an optional multi-endpoint test with Enchytraeus crypticus.

Soil ecotoxicity standard tests for invertebrates are usually limited to the assessment of endpoints like survival and reproduction. Adverse effects may occur at other developmental stages, e.g. embryo development, hatching or growth. The species Enchytraeus crypticus is a model organism in the standard soil ecotoxicology test, where survival and reproduction are assessed. In the present study we optimized the test method to include additional endpoints. The proposed test started with synchronized age organisms', and included additionally hatching success, growth, maturation status and full life cycle. This allowed for the calculation of cocoon production and population growth rate. Results indicated that Cd is embryotoxic, main effect occurs on the embryo developmental stage and maturity. Further, the full life cycle test can discriminate between pre- and post-embryo formation. The increased sensitivity and full life cycle detail level makes it potentially useful for novel materials e.g. nanomaterials where the mode of action and hence effect target is unknown.