Effects of rainbow trout (Oncorhynchus mykiss) cage culture on Western Cape irrigation reservoirs.

Thesis (PhD (Conservation Ecology and Entomology))--University of Stellenbosch, 2011.ENGLISH ABSTRACT: Water storage in reservoirs forms an integral part of the agricultural landscape in the Western Cape Province, South Africa. A few large reservoirs serve primarily as a drinking and industrial water supply, while on private farms, small reservoirs provide irrigation water for the dry summer period. Protection of water quality to secure irrigation and drinking water quality, and the extension of water use efficiency are priority issues in the Western Cape. In the current study, the suitability of rainbow trout (Oncorhynchus mykiss) cage farming as a non-abstractive water use was investigated. The current study concentrated on the identification, and where possible quantification of aquaculture impacts, the identification of successful sites and a description of requirements in which net-cage aquaculture has none or a very low negative impact on water quality (e.g. <15 % change from previous water quality conditions for phosphorus concentrations). In order to study the effects of 5 t trout cage production units in reservoirs <15 ha in area, the general ecology of the irrigation reservoirs was assessed. Sixteen reservoirs without aquaculture production (reference reservoirs) were compared to 26 reservoirs with aquaculture production (production sites with varying production histories). Catchment characteristics were also monitored. Water from different depths (0 m, 2 m, 6 m and near bottom) was tested for physical and chemical qualities as well as nutrient concentrations. Phytoplankton and zooplankton biomass and species composition was monitored. In addition to the general phytoplankton findings, cyanophytes were evaluated for their contribution to algal taint problems that emerged at a number of production sites. Sediments were tested for total phosphorus content and phosphorus release capacity. Indicators and minimum conditions to avoid the most common production problems were formulated. In order to determine long-term production success, which prevents trophic level changes of reservoirs, a mass balance approach (nutrient budget) was employed to indicate the limits for nutrients that can be added. The phosphorus balance indicated long term trends for reservoirs with and without aquaculture. The detailed mass balance approach was compared to a “ready to use” carrying capacity model that estimated the maximum fish load each reservoir could support. The anthropological input of phosphorus into the reservoirs causes a decreasing water quality in the studied reservoirs and this development was also reported for lowland rivers. Twenty percent of the studied reservoirs are in a condition that could be an immediate threat to fish or water bird health (e.g. free ammonia concentrations and pH). Harmful algal blooms were not observed. Aquaculture production evoked changes in water chemistry and ecology in most of the studied reservoirs. Adverse effects of aquaculture sites versus non-aquaculture sites were: increased phytoplankton biomass and species shifts towards sizes >80 μm. The increased phytoplankton abundance influenced pH maxima to values >9 at mid-day. The high pH fluctuations were greatly influenced by the naturally low alkalinity and hence low calcium buffering capacity of Western Cape waters. The deoxygenation of the hypolimnion during stagnation (summer) occurred faster in reservoirs of certain character, greatly dependent on elevation and surface area, with consequent acidification of the hypolimnion, as well as ammonia and total phosphorus (TP) accumulation. In this context, a diversity of each reservoir with and without aquaculture production, with a similar ratio of undisturbed reservoirs to reservoirs with influence of e.g. agriculture, were compared to each other. When grouping the respective differences from the average reference reservoir (i.e. no trout production), a low impact on water quality was manifested at four sites (15 %) with <15 % increase of bottom TP and ammonia, while eight sites (31 %) showed medium effects (59 % mean increase), and a high impact was found at 54 % of the sites (312 % mean increase). In reservoirs without aquaculture, the extent of incoming phosphorus (which could represent an influence by runoff from agricultural land) was very high. However, in small reservoirs (<5 ha), these values were exceeded by the incoming phosphorus from aquaculture practices. In the case of small reservoirs where the carrying capacity was clearly surpassed, effects caused by aquaculture were severe and the assimilation of waste by the system was not possible (in extreme cases aquaculture waste delivered 60 to 90 % of all incoming phosphorus – two to nine times the phosphorus brought in by rivers and runoff). With regards to sediment, only indirect conclusions could be drawn. Aquaculture production increased hypolimnetic anoxia and the latter was shown to increase potential phosphorus release from sediments. This implies that not only will aquaculture increase the phosphorus concentration of surface waters directly, but it will also increase internal loading. The sedimentation rate was increased with cage aquaculture which affects a hypothesized area of approximately 0.2 to 1.0 ha depending on reservoir hydrology. The composition of the sediment increased organic components which can impact on sediment processes. It can be postulated that increased sedimentation of aquaculture waste and extended anoxic conditions impact on macrozoobenthos. Hydrological and nutrient mass exchange of the reservoirs indicated that no annual increase of phosphorus was achieved with low nutrient input (good inflowing water quality) or good water exchange (>5 times per year), and sometimes with extraction of hypolimnetic water during the stagnation period (summer). A model developed by Beveridge (1984) showed similar results to the mass balance approach and can therefore serve as a more ready model to determine suitable stocking rates. The small (man-made) reservoirs in the Western Cape are in a eutrophication process which far exceeds the speed of natural eutrophication (trophic states indicating highly eutrophic or hypertrophic conditions after approximately 10 to 20 years following construction of the reservoirs) and this process is triggered by agricultural practice (indicated by literature – not a subject of this study). However, it is concluded from the data of this study that trout-cage aquaculture duplicated the total phosphorus already present (independent of continuation of the projects, the phosphorus introduced was trapped in the closed systems the reservoirs represent) in only 1 to 2 years of production - which means a significant acceleration of the eutrophication process already in place. There are positive exceptions where trout-cage production is possible without negative effects. Careful site selection is the most important step in successful and sustainable trout production. No impact of aquaculture was recorded at four reservoirs (15 % of the investigated reservoirs) which shared the characteristics of good water exchange (>3 times per year) and a minimum surface area of 5 ha. Additionally, criteria that reduced the risk of algal taint included a minimum water depth of 6 to 7 m in a reservoir at its lowest water point (to avoid intermediate mixing during the stagnation period) as well as cold hypolimnetic conditions (<17 °C) to minimize cyanophyte cyst remobilization. Further improvement of food conversion ratio (feeding management) and feed quality are the next (after site selection) two most important components that determine if a reservoir can be utilised for cage production without any long-term changes. There is potential in advances in feed quality, feed management and waste collection systems. These measures (e.g. the cage size could be decreased to efficate feeding management) can increase the number of sustainable sites and achieve multiplication of water use without water quality deterioration.AFRIKAANSE OPSOMMING: Die stoor van water in reservoirs vorm ‘n integrale deel van die landboulandskap in die Westelike Kaap gedeelte van Suid-Afrika. ‘n Aantal groter reservoirs voorsien primêr water primer vir drinkdoeleindes en vir aanwending deur industrieë, terwyl kleiner reservoirs op private plase gebruik word vir besproeiing gedurende die droë somerperiode. Die beskerming van die waterkwaliteit en die uitbreiding van watergebruik se doeltreffendheid, word bestempel as ‘n prioriteit vir die Wes-Kaap. In die huidige studie is die geskiktheid van forel hokkultuur as ‘n bykomende watergebruik, maar sonder waterverbruik, ondersoek. Die huidige studie fokus op die identifisering en waar moontlik die kwantifisering van die impak van akwakultuur op besproeiingsreservoirs; die identifisering van potensiële reservoirs vir akwakultuur; en die beskrywing van toestande waarin akwakultuur geen of ‘n baie lae negatiewe uitwerking op die waterkwaliteit (bv. < 15 % verandering vanaf oorspronklike waterkwaliteitstoestande vir fosforkonsentrasies) sal hê. Om die impak van 5 t forel hokproduksie eenhede in <15 ha reservoirs te bestudeer, is die algemene ekologie van die besproeiingsreservoirs bepaal. Watermonsters, geneem op verskillende dieptes (0, 2, 6 en naby die bodem), is getoets vir fisiese- en chemiese kwaliteit sowel as vir die nutriëntkonsentrasies. Die biomassa en spesiesamestelling van fitoplankton sowel as zooplankton is gemonitor. Sestien verwysingsreservoirs is vergelyk met 26 produksiereservoirs (met wisselende vlakke van produksiegeskiedenis). Die karakeristieke eienskappe van die opvangsgebied is ook gemonitor. Tesame met die algemene fitoplankton bevindinge, is blougroen alge ook geëvalueer vir hul bydrae tot algae besmettingsprobleme wat voorgekom het by ‘n aantal produksie persele. Sedimente is getoets vir die totale fosforinhoud en fosforvrystellingskapasiteit. Perseelseleksie is geidentifiseer as een van die mees belangrikste faktore vir suksesvolle hokproduksie. Indikatore en minimum toetstande wat vereis word om die mees algemene produksieprobleme te verhoed, is geformuleer. Om die langtermyn produksie sukses te bepaal, wat trophiese vlakke se verandering in die reservoirs verhoed, is ‘n massa balans benadering (nutriënt balans) gebruik. Die fosfor balans het langtermyn tendense aangetoon en in die geval van die produksie reservoirs, is die akwakultuurimpak gekwantifiseer. Die gedetaileerde massa balans benadering is vergelyk met ‘n “gereed om te gebruik” drakapasiteitsmodel wat die maksimum vis wat die reservoir kan onderhou, geskat het. Die antropogeniese toevoer van fosfor na die reservoirs veroorsaak ‘n afname in die waterkwaliteit van die reservoirs wat bestudeer is. Die verskynsel van ‘n afname in waterkwaliteit is ook vir verskeie laagliggende riviere geraporteer. Sowat 20 % van die besproeiingsreservoirs wat bestudeer is, is tans in ‘n toestand wat die onmiddelike gesondheid van vis en watervoëls kan bedreig. Skadelike algebbloei is nie waargeneem nie. Akwakultuurproduksie het negatiewe effekte getoon in ‘n aantal reservoirs. Die nadelige uitwerking van akwakultuur waar digthede te hoog was, is: toename in fitoplankton biomassa en spesiesverskuiwinge. Die toename in fitoplankton teenwoordigheid het die pH maxima beïnvloed tot waardes >9 teen 12 uur middag. Die hoë pH fluktuasies is grootliks beïnvloed deur die natuurlike lae alkaliniteit en gevolglike kalsium bufferkapasitiet van die Wes-Kaap se waterbronne. Die deoksiginasie van die hipolimnion gedurende stagnasie (somer) het vinniger plaasgevind in oorbelaaide reservoirs, met gevolglik ‘n versuring van die hipolymnion, sowel as die akkumelering van ammoniak en totale fosfor. In hierdie konteks word reservoirs met en sonder landbouproduksie, met ‘n gelyke verhouding van onversteurde reservoirs tot reservoirs wat deur landboubedrywighede beinvloed word, met mekaar vergelyk. By vier persele (15 %) is ‘n lae impak vasgestel (<15 % toename in bodem TF en ammoniak), terwyl agt persele (31 %) ‘n medium impak getoon het (59 % gemiddelde toename in bodem TF en ammoniak) en ‘n hoë impak is opgemerk by 54 % van die persele (31 % gemiddelde toename in bodem TF en ammoniak). In reservoirs sonder akwakultuur, was die omvang van fosforinvloeiing baie hoog en is moontlik veroorsaak deur die invloei van afloopwater uit omliggende landbougebiede. Alhoewel in klein reservoirs (<5 ha) was hierdie waardes oorskadu deur die invloeiing van fosfor deur akwakultuur praktyke.In die geval van klein reservoirs, waar die drakrag duidelik oorskry is, was die gevolge, soos veroorsaak deur akwakultuur ernstig, en die assimilasie van afval deur die sisteem nie moontlik nie. In die uiterste gevalle het akwakultuurafval 60 % - 90 % van alle inkomende fosfor gelewer - twee tot nege maal die fosfor wat deur riviere en afloopwater ingevloei het. Wat die sediment aanbetref, kon slegs indirekte gevolgtrekkings gevorm word. Akwakultuurproduksie het hipolimnetiese anoksia laat toeneem en die laasgenoemde verskynsel, het die potensiaal aangedui vir die toename in die vrystelling van fosfor vanaf die sediment. Dit dui daarop dat akwakultuur nie alleen die fosforkonsentrasie in die oppervlaktwater laat toeneem nie, maar sou ook die interne belading laat kon toeneem. Die sedimentasie tempo het toegeneem met die teenwoordigkeid hokkultuur en het ‘n hipotetiese area van ongeveer 0.05 tot 1.00 ha, afhangende van reservoir hidrologie, beïnvloed. Die samestelling van die sediment het toegeneem in teme van die organiese komponente wat die sedimentasie prosesse kon beïnvloed. Dit kan gepostuleer word dat die toename in sedimentasie van akwakultuurafvalprodukte tesame met verlengde anoksiese toestande, ‘n invloed op die makrosoöbentiese organismes het. Hidrologiese en nutriënt massa uitruiling van die reservoirs het aangetoon dat geen jaarlikse toename in fosfor verkry kan word met lae nutrient toelae (kwaliteit van invloeiende water) of met goeie waterverplasing nie, en soms met die ekstraksie van hipoliminetiese water gedurende die stagnasie periode (somer). Die Beveridge model het soortgelyke resultate getoon tot die massabalans benadering en kan daarom dien as ‘n meer aanvaarbare model om gepaste beladingstempo vas te stel. Kleiner mensgemaakte reservoirs in die Wes-Kaap is onderhewig aan ‘n eutrofikasie proses wat die spoed van natuurlike eutrofikasie (trofies verwys na ‘n hoogs eutrofiese of hipertrofiese toestand ongeveer 10 tot 20 jaar na reservoir konstruksie) oorskry. Literatuur (nie ondersoek in die huidige studie) dui aan dat hierdie versnelde eutrofikasie proses meegebring word deur landbouaktiwiteite in die opvangsarea van die reservoirs. Resultate van die huidige studie het getoon dat forelproduksie in hokstelsels, die konsentrasie van total fosfor wat reeds beskikbaar was, verdubbel het. Die toename in fosforkonsentrasie het binne 1 tot 2 jaar na die aanvang van forelproduksie in die betrokke reservoirs, plaasgevind. Daar is egter uitsonderings waar forelproduksie in hokstelsels moontlik was, sonder die gepaardgaande afname in die waterkwaliteit. Die belangrikste stap vir suksesvolle en volhoubare forelproduksie is deeglike perseelseleksie. Daar is geen impak van akwakultuur waargeneem by vier persele (15 %) wat die eienskappe van goeie waterverplasing (>3 keer per jaar) en ‘n minimum oppervlakarea van 5 ha gehad het nie. Bykomend, sluit kriteria wat die risiko van algbesmetting laat afneem, ‘n minimum waterdiepte van 6 tot 7 m in ‘n reservoir by die laagste punt in (om te verhoed dat intermediêre vermenging plaasvind gedurende die stagnasieperiode) sowel as koue hipolimnetiese toestande (<17 °C) om sianobakterieë sist remobilisasie te minimaliseer. Verdere verbetering van die VOH (voeromsettingsverhouding onder voedingsbestuur) en voerkwaliteit is na perseeleleksie, die volgende komponente wat kan aandui of ‘n perseel gebruik kan word vir hokkultuur sonder enige impak. Vordering met voerkwaliteit en voedingsbestuur kan die aantal volhoubare persele laat toeneem en daardeur meer effektiewe watergebruik teweeg bring, sonder die verwante waterkwaliteit verswakking

Ste7 variants that promote pathway specificity in S. cerevisiae

The yeast S. cerevisiae must respond to a wide variety of external stimuli with the appropriate differential program to ensure survival. Yeast cells transmit signals from external environmental cues via signaling cascades to bring about proper transcriptional programs. One such signaling cascade, the Mitogen Activated Protein Kinase (MAPK) cascade, is conserved in all organisms. The MAPK cascade that contains the sequentially acting kinases Ste20, Ste11, Ste7 and Kss1 is used in both filamentous growth differentiation in response to limiting nutrients as well as differentiation in response to mating pheromone. Despite the shared components signaling fidelity remains. The central kinase in both the mating and filamentous growth differentiation pathways is Ste7. We used a constitutively active version of this kinase to examine if biological specificity is lost by removing the need for external stimulus. We found that the constitutively active Ste7 promoted filamentous growth while simultaneously failing to support mating differentiation. One version of constitutively active Ste7 that could not be feedback phosphorylated in a MAPK dependent manner was unable to bind to the MAPK Fus3 but retained the ability to bind to Ste5 and Kss1. Despite being able to bind to Ste5 this version of Ste7 was unable to activate Fus3 but retained the ability to activate Kss1. The activation of Kss1 by constitutively active Ste7 is independent of the scaffold protein Ste5. Unlike Kss1, Fus3 activation requires Ste5. We isolated a substitution mutation variant of Ste7 that was impaired for binding to Ste5 but not impaired for activation of the transcriptional reporter gene for filamentous growth. This variant of Ste7 neither activated Fus3 nor supported mating differentiation in response to pheromone. Yeast cells expressing this variant of Ste7 displayed higher levels of phosphorylated Mpk1, the MAPK that functions in response to plasma membrane distortions. Our findings uncovered a role for Ste5 in maintaining signal specificity by allowing for specific activation of Fus3 by Ste7 in response to mating pheromone. We also uncovered a novel role that Mpk1 may play in the transcriptional response during filamentous growth. These data suggest MAPK signaling occurs through a network rather than linear pathways

Service robotics: do you know your new companion? Framing an interdisciplinary technology assessment

Service-Robotic—mainly defined as “non-industrial robotics”—is identified as the next economical success story to be expected after robots have been ubiquitously implemented into industrial production lines. Under the heading of service-robotic, we found a widespread area of applications reaching from robotics in agriculture and in the public transportation system to service robots applied in private homes. We propose for our interdisciplinary perspective of technology assessment to take the human user/worker as common focus. In some cases, the user/worker is the effective subject acting by means of and in cooperation with a service robot; in other cases, the user/worker might become a pure object of the respective robotic system, for example, as a patient in a hospital. In this paper, we present a comprehensive interdisciplinary framework, which allows us to scrutinize some of the most relevant applications of service robotics; we propose to combine technical, economical, legal, philosophical/ethical, and psychological perspectives in order to design a thorough and comprehensive expert-based technology assessment. This allows us to understand the potentials as well as the limits and even the threats connected with the ongoing and the planned implementation of service robots into human lifeworld—particularly of those technical systems displaying increasing grades of autonomy

Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites

Synthesis of earthworm trace metal uptake and bioaccumulation data: role of soil concentration, earthworm ecophysiology, and experimental design

Trace metals can be essential for organo-metallic structures and oxidation-reduction in metabolic processes or may cause acute or chronic toxicity at elevated concentrations. The uptake of trace metals by earthworms can cause transfer from immobilized pools in the soil to predators within terrestrial food chains. We report a synthesis and evaluation of uptake and bioaccumulation empirical data across different metals, earthworm genera, ecophysiological groups, soil properties, and experimental conditions (metal source, uptake duration, soil extraction method). Peer-reviewed datasets were extracted from manuscripts published before June 2019. The 56 studies contained 3513 soil-earthworm trace metal concentration paired data sets across 11 trace metals (As, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Sb, U, Zn). Across all field and laboratory experiments studied, the median concentrations of Hg, Pb, and Cd in earthworm tissues that were above concentrations known to be hazardous for consumption by small mammals and avian predators but not for Cu, Zn, Cr, Ni, and As. Power regressions show only Hg and Cd earthworm tissue concentrations were well-correlated with soil concentrations with R2 > 0.25. However, generalized linear mixed-effect models reveal that earthworm concentrations were significantly correlated with soil concentrations for log-transformed Hg, Cd, Cu, Zn, As, Sb (p < 0.05). Factors that significantly contributed to these relationships included earthworm genera, ecophysiological group, soil pH, and organic matter content. Moreover, spiking soils with metal salts, shortening the duration of exposure, and measuring exchangeable soil concentrations resulted in significantly higher trace metal uptake or greater bioaccumulation factors. Our results highlight that earthworms are able to consistently bioaccumulate toxic metals (Hg and Cd only) across field and laboratory conditions. However, future experiments should incorporate greater suites of trace metals, broader genera of earthworms, and more diverse laboratory and field settings to generate data to devise universal quantitative relationships between soil and earthworm tissue concentrations

The ability of terrestrial Oligochaeta to survive in ultramafic soils and the assessment of toxicity at different levels of organisation

Thesis (PhD (Botany and Zoology)) -- University of Stellenbosch, 2006.Metals are natural elements of the earth crust usually present at low concentrations in all soils. Although many metals such as cobalt, copper, iron and zinc are essential to living organisms, at elevated concentrations most metals are toxic to organisms living in and on soils. Elevated concentrations of metals are caused either by anthropogenic deposition following remobilisation from the earth crust or are of natural origin. Ultramafic soils do not only pose unfavourable living conditions such as drought and poor organic content, these soils are also characterized by extremely high concentrations of a range of metals known to be toxic under normal circumstances. Ultramafic soils are of high ecological importance as a high proportion of endemic organisms, especially plants, live on these soils. As it is known that earthworms do occur in ultramafic soils, the aims of the present study were to investigate the abilities of earthworms to survive in these soils and the influences of elevated chromium, cobalt, copper, manganese and nickel levels. For the evaluation of the metal background conditions, soils originating from ultramafic rocks of the Barberton Greenstone Belt, Mpumalanga, South Africa were collected and different fractions representing different levels of bioavailability were analyzed for arsenic, chromium, cobalt, copper, manganese and nickel. To assess the mobile, readily available metal fraction, i.e. Ca2+- exchangeable metal cations, a 0.01 mol/L CaCl2 extraction was performed. To investigate the mobilisable metal fraction, representing the amount of easily remobilisable complexed and carbonated metal ions, a DTPA (di-ethylene-triamine-pentaacetic acid) extraction was conducted. In relation to non-ultramafic or anthropogenic contaminated soils, a far lower proportion of metals were extractable by the above mentioned extraction methods. To investigate the availability and effects of these metals on earthworms, two ecophysiologically different species were employed. Aporrectodea caliginosa and Eisenia fetida were long-term exposed to the ultramafic soils collected at the Barberton region and a control soil from a location at Stellenbosch with a known history of no anthropogenic metal contamination. The responses to the ecological stress originating in the ultramafic soils were measured on different levels of earthworm organisation. As endpoints affecting population development, cocoon production, fecundity and viability were evaluated. On individual level, growth, metal body burden and tissue distribution were investigated. As endpoints on subcellular level, the membrane integrity was assessed by the neutral red retention assay, the mitochondrial activity was measured by the MTT colorimetric assay and as a biomarker for the DNA integrity, the comet assay was performed. Focussing on manganese and nickel, the uptake by E. fetida of these metals was investigated with the exclusion of soil related properties using an artificial aqueous medium to draw comparisons to the uptake of these metals in natural soils. The possible development of resistance towards nickel was tested by exposing pre-exposed (for more than 10 generations) E. fetida specimens to ultramafic soils with concentrations of more than 4000 mg/kg nickel. The results showed that, except on the endpoint survival, which was less sensitive than all other bioassays, significant responses to the ultramafic challenge were observed in all earthworm bioassays and on all levels of organisation. The sensitivity of the responses of the earthworms towards the ultramafic conditions was not predictable by the level of organisation. The two species showed different strategies of metal elimination. In A. caliginosa, metals such as nickel, manganese and chromium were transported to the posterior section and the posterior section was subsequently pushed off by autotomization. In E. fetida, metals such as chromium and nickel were sequestered in storage compartments in the coelomic cells or fluid. Other metals, such as cobalt, were not taken up at elevated concentrations. Although an increased accumulation of nickel was observed in E. fetida specimens pre-exposed to nickel, development of resistance or cross resistance was not observed in this species. In contrast, pre-exposed specimen exposed to elevated concentrations of nickel showed a higher sensitivity in terms of survival, indicating the absence of acclimatisation or even genetic adaptation. A comparison of the two species employed indicated that A. caliginosa was less suited for the assessment of the ultramafic soils due to the high individual variation in metal body burden, the mass loss observed and the slow reproduction rate even in the control soils. This happened despite the fact that A. caliginosa was a soil dwelling species supposed to be better adapted to the soil substrate than the litter dwelling E. fetida. The toxicity of the ultramafic soils was not necessarily related to total or environmentally available amounts of the selected metals. Thus, it can be speculated that either these soils contained unidentified toxicants with resulting interactions between toxicants playing an important role or earthworms were able to remobilize metals occurring in these soils. As the singular application of an ecotoxicological endpoint did not give reliable results, especially seen over the duration of the exposures, it can be concluded that, when studying soils with such a complex composition, the utilisation of endpoints addressing different levels of organisation is necessary for the assessment of toxic stress emerging from these ultramafic soils