Parasite infection influences the biomarker response and locomotor activity of Gammarus fossarum exposed to conventionally-treated wastewater.

Modern wastewater treatment plants cannot completely remove pollutants. Often, effluents entering the aquatic environment still contain micropollutants such as pharmaceuticals or pesticides, which may impose adverse effects on aquatic biota. At the same time, a large proportion of free-living aquatic species are known to be infected with parasites, which raises the question of interactions between environmental stressors (such as micropollutants) and parasite infection. We chose the freshwater amphipod Gammarus fossarum (Koch, 1835) as a test organism to investigate potential pollutant-parasite interactions. This gammarid is frequently used in ecotoxicological tests and is also commonly infected with larvae of the acanthocephalan parasite species Polymorphus minutus (Zeder, 1800) Lühe, 1911. We exposed infected and uninfected specimens of G. fossarum to conventionally-treated wastewater and river water in a 22-day flow channel experiment. The test organisms' response was measured as mortality rates, concentrations or activities of five biomarkers, and overall locomotor activity. No significant differences were found between mortality rates of different exposure conditions. Contrastingly, three biomarkers (phenoloxidase activity, glycogen, and lipid concentrations) showed a significant increase in infected gammarids, while the effect of the water type was insignificant. Infected gammarids also showed a significantly higher locomotor activity in both water types. Our results suggest that the response of G. fossarum during the exposure experiments was mainly driven by parasite infection. This implies that parasites may act as additional biotic stressors in multiple stressor scenarios, and therefore, might play an important role when measuring the response of organisms to chemical stressors. Future ecotoxicological studies and assessments thus should consider parasite infection as an additional test parameter.

Use of the Multispecies Freshwater Biomonitor to assess behavioral changes of Poecilia reticulata (Cyprinodontiformes: Poeciliidae) and Macrobrachium lanchesteri (Decapoda: Palaemonidae) in response to acid mine drainage: laboratory exposure.

The behavioral responses of guppy Poecilia reticulata (Poeciliidae) and prawn Macrobrachium lanchesteri (Palaemonidae) individuals exposed to acid mine drainage (AMD) were monitored online in the laboratory with a Multispecies Freshwater Biomonitor™ (MFB). These responses were compared to those to reference water acidified to the respective pH values (ACID). Test animals in the juvenile stage were used for both species and were exposed to AMD and ACID for 24 hours. The stress behaviors of both test animals consisted mainly of decreased activity in AMD and increased activity in ACID, indicating that the metals in the AMD played a role as a stress factor in addition to pH. The locomotor activity levels of guppies and prawns for the ACID treatment were higher than the locomotor activity levels for the AMD treatment with increasing pH value. For guppies, significant differences were observed when specimens were exposed to AMD and ACID at pH 5.0 and 6.0; the percentage activities were only 16% and 12%, respectively, for AMD treatment, whereas for ACID treatment, the percentage activities were 35% and 40%, respectively, similar to the value of 36% for the controls. Similar trends were also observed for prawns, for which the percentage activities were only 6% and 4%, respectively, for AMD treatment, whereas for ACID treatment, the percentage activities were 31% and 38%, respectively, compared to 44% in the controls. This study showed that both species are suitable for use as indicators for ecotoxicity testing with the MFB.

Freshwater systems and ecosystem services: Challenges and chances for cross-fertilization of disciplines.

Freshwater ecosystems are among the most threatened in the world, while providing numerous essential ecosystem services (ES) to humans. Despite their importance, research on freshwater ecosystem services is limited. Here, we examine how freshwater studies could help to advance ES research and vice versa. We summarize major knowledge gaps and suggest solutions focusing on science and policy in Europe. We found several features that are unique to freshwater ecosystems, but often disregarded in ES assessments. Insufficient transfer of knowledge towards stakeholders is also problematic. Knowledge transfer and implementation seems to be less effective towards South-east Europe. Focusing on the strengths of freshwater research regarding connectivity, across borders, involving multiple actors can help to improve ES research towards a more dynamic, landscape-level approach, which we believe can boost the implementation of the ES concept in freshwater policies. Bridging these gaps can contribute to achieve the ambitious targets of the EU's Green Deal.

Gammarus spp. in aquatic ecotoxicology and water quality assessment: toward integrated multilevel tests.

The amphipod genus Gammarus is widespread and is structurally and functionally important in epigean freshwaters of the Northern Hemisphere. Its presence is crucial, because macroinvertebrate feeding is a major rate-limiting step in the processing of stream detrius. In addition, Gammarus interacts with multiple trophic levels bu functioning as prey, predator, herbivore, detritivore, and shredder. Such a broad span of ecosystem participation underlines the importance of Gammarus to pollutants and other disturbances may render it a valuable indicator for ecosystem health. This review summarizes the vast number of studies conducted with Gammarus spp. for evaluating aquatic ecotoxicology endpoints and examines the suitability of this native invertabrate species for the assessment of stream ecosystem health in the Northern Hemisphere. Numerous papers have been published on how pollutants affect gammarind behavior (i.e., mating, predator avoidance), reproduction, development, feeding activity, population structure, as well as the consequences of pollution on host-parasite, predator-prey, or native-invasive species interactions. Some biochemical and molecular biomarkers have already been established, such as the measurement of vitellogenin-like proteins, metallothioneins, alkali-labile phosphates (in proteins), and lipogenic enzyme activities for assessing endocrine distribution and detoxification mechanisms.

Locomotor activity and respiration rate of the ground beetle, Pterostichus oblongopunctatus (Coleoptera: Carabidae), exposed to elevated nickel concentration at different temperatures: novel application of Multispecies Freshwater Biomonitor.

The aim of the work was to study effects of suboptimal temperature on nickel toxicity under chronic exposure and to apply the Multispecies Freshwater Biomonitor (MFB) for the first time to soil organisms in order to link behavioral to physiological endpoints. Ground beetles Pterostichus oblongopunctatus were reared at 10 or 20 degrees C on control or Ni-contaminated (2500 mg Ni kg(-1)) food. After 64 days half of the Ni-exposed beetles were transferred to uncontaminated food (elimination phase). The remaining Ni-exposed beetles were left on the contaminated food to the end of the experiment (96 days). After completing the experiment, respiration rate and locomotor activity were measured in Ni-contaminated beetles (Ni), Ni-contaminated ones after elimination (E), and controls (C). Then, the beetles were analyzed for Ni body loads. The respiration rate, which was always measured at 20 degrees C for all experimental groups, was highest in Ni beetles reared at 10 degrees C and did not differ between groups C and E. Similarly, locomotor activity was highest in Ni beetles, and marginally significant temperature effect was found. The study indicated thus that exposure to elevated Ni concentrations increased the maintenance costs in P. oblongopunctatus and that rearing the beetles at suboptimal temperature increased the respiration rate even further. However, the effects observed in both respiration rate and locomotor activity were reversible after decontamination. The study demonstrated also the potential of MFB for assessing the behavior of soil-dwelling organism in environmental toxicology.

Impairment of trophic interactions between zebrafish (Danio rerio) and midge larvae (Chironomus riparius) by chlorpyrifos.

The effects of chemicals on biotic interactions, such as competition and predation, have rarely been investigated in aquatic ecotoxicology. This study presents a new approach for the investigation of predator-prey interactions between zebrafish (Danio rerio) and midge larvae (Chironomus riparius) impaired by chlorpyrifos (CHP), a neurotoxic insecticide. With a simple experimental design including four different treatments: (1) control, (2) predator exposed, (3) prey exposed and (4) both, predator and prey, exposed, we were able to detect by visual observation an increase in the feeding rate of zebrafish preying on exposed chironomids after acute (2 h) exposure to 6 µg/l CHP. Previously, a decrease in the burrowing behaviour of exposed chironomid larvae was observed. However, when pre-exposing simultaneously both predators and prey, no significant differences in the feeding rate of zebrafish were observed. This suggests an impairment in prey recognition of the exposed zebrafish. At a lower CHP concentration (1 µg/l), no differences in feeding rate of zebrafish were observed. We therefore propose the use of trophic interactions as parameters in higher tier studies for chemical testing and evaluation of ecotoxicological risk assessment.

Assessing toxicity of the insecticide thiacloprid on Chironomus riparius (Insecta: Diptera) using multiple end points.

Since data documentation on neonicotinic toxicity to nontarget organisms should be enhanced, we investigate the effects of thiacloprid, a novel neonicotinoid insecticide, on the sediment-dwelling nontarget insect Chironomus riparius. Further, we wanted to validate the sensitivity of end points on different biological levels and obtain the greatest amount of information regarding the effects of this compound by using a battery of several end points such as larval mortality, behavior, body weight gain, emergence rate, time of development, gender ratio, Hsp70 stress protein level, and larval mouthpart deformities after exposure at a concentration range of 0.1 to 1000 microg/L thiacloprid. C. riparius was impacted starting at concentrations of 0.5 microg/L, a concentration that can be considered environmentally relevant. Larval mortality, behavior, emergence, and Hsp70 protein level were sensitive indicators for the toxic effect of thiacloprid, whereas gender ratio and mouthpart morphology were not affected. In our case life-cycle end points like survival rate (LC(50): 1.57 microg/L) and emergence rate (EC(50): 0.54 microg/L) proved to be more sensitive than tested physiological end points for the neurotoxic insecticide.

Assessment of the Multispecies Freshwater Biomonitor (MFB) in a marine context: the Green crab (Carcinus maenas) as an early warning indicator.

The Multispecies Freshwater Biomonitor is an online continuous biomonitor which utilises impedance conversion to quantitatively record behavioural responses of vertebrates and invertebrates to environmental change. Here, we extend the use of the MFB into the marine aquaculture environment using the Green crab (Carcinus maenas) as a biological monitor. As a ubiquitous and abundant species, C. maenas can be used in applications such as aquaculture and monitoring of diffuse and point source marine pollution. Four experiments were undertaken to establish: (1) if the electrical field generated by the apparatus had any effect on C. maenas; (2) if the behaviour of C. maenas was altered by the presence of ammonia; (3) if the behaviour of C. maenas was affected by the electrical field when ammonia was present and (4) if defined behaviours could be detected by the MFB. There was no significant effect of the current on C. maenas in the MFB. There was a significant difference in overall expression of behaviour in response to an increasing gradient of ammonia and activity of the chamber. Five behaviours, 'walking', 'climbing', 'leg stretch', 'cleaning' and 'inactivity' were detected by the MFB. C. maenas appears to be a suitable candidate for use in the MFB in a marine context. Further testing of the biomonitor and C. maenas is required using other toxicants to establish alarm thresholds that could be used in situ for water quality monitoring.

Behavioural and developmental toxicity of chlorpyrifos and nickel chloride to zebrafish (Danio rerio) embryos and larvae.

In order to assess the combined toxicity of environmental chemicals with different modes of action in acute (2 h) and subchronic (11 d) exposures, embryos and larvae of Danio rerio were exposed to a heavy metal salt, nickel chloride (NiCl2), the insecticide chlorpyrifos (CHP) and their binary mixtures. Chlorpyrifos is an acetylcholine esterase inhibitor, which is likely to affect behaviour of the organism. NiCl2 targets the active sites of enzymes and is regarded as an unspecific toxicant for aquatic organisms. Several endpoints, such as locomotor activity, morphological abnormalities, and mortality of D. rerio embryos and larvae were studied. During acute exposures to > or =0.25 mg/L of chlorpyrifos, locomotor activity tended to increase. However, this activity decreased significantly at > or =7.5 mg Ni/L. Subchronic exposures to CHP resulted in behavioural changes at much lower concentrations (> or =0.01 mg/L) and considerably earlier than the observed increase in morphological abnormalities and mortality (LC(50) (10 d): 0.43 mg/L). Combined CHP and NiCl2 mixtures led to an antagonistic deviation from the concept of independent action, in the case of locomotor activity. Compared to developmental or survival parameters, behaviour was the most sensitive endpoint for CHP exposure in this study; therefore we recommend this parameter to complement already established endpoints.

Effects of 3,4-dichloroaniline and diazinon on different biological organisation levels of zebrafish (Danio rerio) embryos and larvae.

In this study the effects of 3,4-dichloroaniline (3,4-DCA), a decomposition product of the herbicides propanil and diuron (and other pesticides), and diazinon, a neurotoxic insecticide, on early life stages of zebrafish Danio rerio were assessed. The toxicity of these substances with different modes of action (acetylcholine esterase inhibitor vs. polar narcosis) was tested for single substances as well as in binary mixtures. To study effects on different biological organisation levels (from the molecular up to the whole organism level) the molecular stress response regarding Hsp70, the embryonic and larval development and the locomotor activity were investigated as integrative biomarkers. In single substance tests 3,4-dichloroaniline elicited deformations at > or = 0.25 mg/l during the 11 days subchronic test, whereas locomotor activity and mortality were impaired at > or = 0.5 mg/l. Diazinon effects on those parameters were obvious at > or = 2 mg/l, except for the deformation rate (11 days:1 mg/l). In equitoxic mixtures of both substances concentration additivity was observed for deformation rate and mortality (11 days). An increase in the Hsp70 content occurred in zebrafish exposed to 0.25 mg 3,4-DCA/l as well as to 0.05 mg diazinon/l; in mixtures concentration additivity could be shown. The investigated endpoints varied in respect to their sensitivity, with deformations and Hsp70 levels as most sensitive parameters concerning 3,4-DCA and Hsp70 as most sensitive parameter concerning diazinon. Accordingly, for an integrated understanding of the effects of chemicals and their mixtures on fish, a battery of different test methods should be applied.

Effects of nickel chloride and oxygen depletion on behaviour and vitality of zebrafish (Danio rerio, Hamilton, 1822) (Pisces, Cypriniformes) embryos and larvae.

We examined acute (2 h exposure of 5-day-old larvae) and subchronic (exposure from fertilization up to an age of 11 days) effects of NiCl(2).6H2O on embryos and larvae of zebrafish (Danio rerio), both alone and in combination with oxygen depletion. The following endpoints were recorded: acute exposure: locomotory activity and survival; subchronic exposure: hatching rate, deformations, locomotory activity (at 5, 8 and 11 days) and mortality. In acute exposures nickel chloride (7.5-15 mg Ni/L) caused decreasing locomotory activity. Oxygen depletion (or=10 mg Ni/L resulted in delayed hatching at an age of 96 h, in decreased locomotory activity at an age of 5 days, and increased mortality at an age of 11 days (LC20=9.5 mg Ni/L). The observed LOEC for locomotory activity (7.5 mg Ni/L) is in the range of environmentally relevant concentrations. Since locomotory activity was already affected by acute exposure, this parameter is recommended to supplement commonly recorded endpoints of toxicity.

Behavior of Corophium volutator (Crustacea, Amphipoda) exposed to the water-accommodated fraction of oil in water and sediment.

We investigated the short-term effects of the water accommodated fraction (WAF) of weathered Forties crude oil on the behavior of Corophium volutator in the Multispecies Freshwater Biomonitor (MFB). When exposing C. volutator to 25 and 50% WAF in aqueous exposures, hyperactivity with an additional increase in ventilation was detected, whereas exposure to 100% WAF led to hypoactivity (narcosis). In a sediment exposure with 100% WAF, there was an increased tendency toward hyperactivity. In a pulse experiment, hyperactivity appeared at and after a 130-min exposure to 50% WAF in a majority of cases. Our experiments suggest that the behavior of C. volutator as measured in the MFB may be an appropriate parameter for coastal monitoring.

Behavioural and feeding responses of Echinogammarus meridionalis (Crustacea, Amphipoda) to acid mine drainage.

As a result of mining activities the exposure of metal sulphides to oxidation takes place with consequent release of acid mine drainage (AMD). Biomonitoring instruments have been proven to have the best deterrent effect upon polluters. A new approach in online biomonitoring, with the Multispecies Freshwater Biomonitortrade mark (MFB), was developed combining behavioural and feeding responses of the Portuguese indigenous benthic shredder, Echinogammarus meridionalis (Pinkster, 1973) (Crustacea, Amphipoda). These endpoints, along with mortality, were measured and analyzed for a gradient of sublethal doses of AMD. Original river water was used as the control and three doses of treatments were attained by adding increasing volumes of AMD to the control. The increase in AMD concentration and concomitant decrease in pH and increase in the concentration of most metals was followed by an overall increase of the mortality, decrease of locomotion/feeding activity and inhibition of the feeding rate. Mortality was observed in the two highest concentrations of AMD. Significant decrease in average locomotion/feeding activity took place in the second treatment. Although an inhibition of feeding was observed along the gradient of AMD concentration only in the highest concentration the feeding rate was significantly reduced.

Importance of exposure route for behavioural responses in Lumbriculus variegatus Müller (Oligochaeta: Lumbriculida) in short-term exposures to Pb.

GOAL, SCOPE AND BACKGROUND: Lumbriculus variegatus Miller (Oligochaeta), a common freshwater sediment-dweller, has frequently been used in toxicokinetic studies, although has been less used in ecotoxicity tests. METHODS: For the first time the Multispecies Freshwater Biomonitor (MFB) was applied in a short-term whole-sediment toxicity test. The MFB automatically and quantitatively recorded the spontaneous locomotory behaviour of Lumbriculus variegatus in exposures with two compartments, water and sediment. The study questioned, whether the animals altered their locomotion depending on the compartment which was spiked with lead (Pb). RESULTS AND DISCUSSION: As in the exposures to Pb-contaminated water/clean sediment, the animals exposed to Pb-contaminated sediment/clean water showed higher activities in intermediate Pb-concentrations. This indicates, that spontaneous locomotory activity is affected by Pb-concentrations at sublethal levels regardless of whether the Pb-concentration is found in the water or in the sediment, because these animals use both environmental compartments simultaneously. However, within the same Pb-levels, the animals showed higher locomotory activity in contaminated water compared with contaminated sediment. This indicates a possible tendency to withdraw from ('avoidance') contaminated water into the clean sediment compartment, whereas there was no withdrawal from contaminated sediment into clean water. The latter might be explained by the fact that withdrawal from sediment to water might increase the risk of predation and drift in nature, whereas retracting to sediment might provide shelter, CONCLUSIONS: The study showed that spontaneous locomotory responses of L. variegatus to Pb depend on whether the water or sediment is contaminated. The study also concluded that the Multispecies Freshwater Biomonitor can be applied effectively in sediment toxicity testing. RECOMMENDATIONS AND PERSPECTIVES: More emphasis should be given to the interactions of water/sediment in sediment ecotoxicity tests to better simulate field conditions and increase ecological realism in risk assessment, especially as quantitative recording methods exisit.

Assessing ecotoxicity of biomining effluents in stream ecosystems by in situ invertebrate bioassays: A case study in Talvivaara, Finland.

Mining of sulfide-rich pyritic ores produces acid mine drainage waters and has induced major ecological problems in aquatic ecosystems worldwide. Biomining utilizes microbes to extract metals from the ore, and it has been suggested as a new sustainable way to produce metals. However, little is known of the potential ecotoxicological effects of biomining. In the present study, biomining impacts were assessed using survival and behavioral responses of aquatic macroinvertebrates at in situ exposures in streams. The authors used an impedance conversion technique to measure quantitatively in situ behavioral responses of larvae of the regionally common mayfly, Heptagenia dalecarlica, to discharges from the Talvivaara mine (Sotkamo, Northern Finland), which uses a biomining technique. Behavioral responses measured in 3 mine-impacted streams were compared with those measured in 3 reference streams. In addition, 3-d survival of the mayfly larvae and the oligochaete Lumbriculus variegatus was measured in the study sites. Biomining impacts on stream water quality included increased concentrations of sulfur, sulfate, and metals, especially manganese, cadmium, zinc, sodium, and calcium. Survival of the invertebrates in the short term was not affected by the mine effluents. In contrast, apparent behavioral changes in mayfly larvae were detected, but these responses were not consistent among sites, which may reflect differing natural water chemistry of the study sites. Environ Toxicol Chem 2017;36:147-155. © 2016 SETAC.

In situ on-line toxicity biomonitoring in water: recent developments.

In situ on-line biomonitoring is an emerging branch of aquatic biomonitoring. On-line biomonitoring systems use behavioral and/or physiological stress responses of caged test organisms exposed in situ either in a bypass system or directly in-stream. Sudden pollution waves are detected by several existing single-species on-line biomonitors, which until now have been placed mostly in streamside laboratories. However, recent achievements have been multispecies biomonitors, mobile biomonitors for direct in-stream use, development of new instruments, new methods for data analysis and alarm generation, biomonitors for use in soil and sediment, and scientific research supporting responses as seen in on-line biomonitors by linking them to other biological and ecological effects. Mobile on-line monitoring platforms containing an array of biomonitors, biosensors, and chemical monitoring equipment might be the future trend, especially in monitoring transboundary rivers at country borders as well as in coastal zones.

Suitability of Crangonyx pseudogracilis (Crustacea: Amphipoda) as an early warning indicator in the multispecies freshwater biomonitor.

BACKGROUND: Biological monitors are increasingly important in 'Biological Early Warning Systems' (BEWS) for monitoring water quality. This study examines the freshwater amphipod Crangonyx pseudogracilis as a potential new indicator species when used in the Multispecies Freshwater Biomonitor (MFB). The MFB is an online continuous biomonitor which uses impedance conversion to record behavioural responses of vertebrates and invertebrates. METHODS: Four experiments were undertaken to establish: (1) if the electrical field generated by the MFB affected the organisms' behaviour, (2) if defined behaviours and their response to a gradient of ammonium chloride could be detected by the MFB, (3) if there was variation in the behaviour of C. pseudogracilis over a diel cycle, and (4) if behaviour changed significantly in response to a pulse of ammonium chloride. RESULTS AND DISCUSSION: Results showed no significant effect of the MFB's current on behaviour of C. pseudogracilis. Four behaviours; swimming, walking, grooming and inactivity, were observed and identified in the MFB. In the MFB, each behaviour changed significantly in response to an increasing gradient of ammonium chloride exposure. The MFB also detected increases in nocturnal activity by C. pseudogracilis. The MFB also detected a significant increase in activity after a pulse of ammonium chloride. CONCLUSION: The range of behaviours exhibited by Crangonyx pseudogracilis, together with its ease of culture, suggest future potential of this species as an indicator species for the Multispecies Freshwater Biomonitor. RECOMMENDATIONS AND OUTLOOK: Further testing is required over a range of toxicants and concentration gradients to establish threshold responses and the full compliment of behaviours that could be useful in online biomonitoring.

Measurement of movement patterns of Caenorhabditis elegans (Nematoda) with the Multispecies Freshwater Biomonitor (MFB)--a potential new method to study a behavioral toxicity parameter of nematodes in sediments.

The nematode Caenorhabditis elegans receives increasing attention in sediment ecotoxicology and new toxicity tests with sensitive test parameters are under development. In this study, the motility of C. elegans could be measured for the first time online in sediment, using the Multispecies Freshwater Biomonitor. Whereas single nematodes could not be recorded, groups of 10 nematodes gave typical locomotive signals in different media (water, agar, sediment) with comparable precision and accuracy. The results of this study encourage to develop a new rapid online whole-sediment toxicity test with behaviour as sensitive test parameter.

Effects of acid mine drainage on larval Chironomus (Diptera, Chironomidae) measured with the Multispecies Freshwater Biomonitor.

The abandoned Säo Domingos mine (Portugal) offers a pH and metal gradient of acid mine drainage (AMD), and is an ideal model for ecotoxicological studies. Short-term 24-h bioassays with water from the AMD (pH 3.3, 4.4, and 5.5, and control) were performed with fourth instars of Chironomus in the laboratory and in situ (AMD at pH 5.5) in artificial flow-through channels. This was compared to reference water acidified to the respective pH values (acid only). Behavioral stress responses were monitored online with the Multispecies Freshwater Biomonitor (MFB). The exposure to AMD and acid only was in the sublethal range (mortality 0-20%). The use of MFB chambers did not affect survival. Stress behavior of Chironomus consisted mainly of decreased locomotory activity in AMD and increased activity in acid-only tests, indicating that the metals in the AMD played a role as stress factor. Field exposure in the AMD mixing zone (pH 5.5) generated similar activity as in the corresponding laboratory exposure.

Effect of the exposure to metal lead on the regenerative ability of Lumbriculus variegatus (Oligochaeta).

Lumbriculus variegatus is a recommended species for use in sediment toxicity tests and is known to have a remarkable power of segmental regeneration. Here, we tested the effects of a chemical stressor on the regenerative ability of L. variegatus and investigated the potential of regenerative ability as an additional new parameter in standard toxicity tests. The worms were cut into two equal segments, and exposed to various concentrations of lead. Two assays were performed: one with sediment spiked with lead and the other with water spiked with lead. The endpoints were segmental regeneration, survival and behaviour. Regenerative ability was clearly affected by exposure to lead-contaminated sediment and lead-contaminated water. Organisms exposed to lead grew more slowly than those not exposed; worms exposed to contaminated water showed higher mortalities than those exposed to contaminated sediment. Results showed that L. variegatus' regenerative ability, as a developmental test parameter, is more sensitive than mortality.