Species-specific differences and temperature-dependence of Na+/K+-ATPase in freshwater mussels Anodonta anatina and Unio tumidus (Bivalvia: Unionidae).

The predicted global warming of surface waters can be challenging to aquatic ectotherms like freshwater mussels. Especially animals in northern temperate latitudes may face and physiologically acclimate to significant stress from seasonal temperature fluctuations. Na+/K+-ATPase enzyme is one of the key mechanisms that allow mussels to cope with changing water temperatures. This enzyme plays a major role in osmoregulation, energy control, ion balance, metabolite transport and electrical excitability. Here, we experimentally studied the effects of temperature on Na+/K+-ATPase activity of gills in two freshwater mussel species, Anodonta anatina and Unio tumidus. The study animals were acclimated to three ambient temperatures (+4, +14, +24 °C) and Na+/K+-ATPase activity was measured at those temperatures for each acclimation group. Both species had their highest gill Na+/K+-ATPase activity at the highest acclimation temperature. Na+/K+-ATPase activity of gills exhibited species-specific differences, and was higher in A. anatina than U. tumidus in all test groups at all test temperatures. Temperature dependence of Na+/K+-ATPase was confirmed in both species, being highest at temperatures between +4 and + 14 °C when Q10 values in the acclimation groups varied between 5.06 and 6.71. Our results underline the importance of Na+/K+-ATPase of gills for the freshwater mussels in warming waters. Because Na+/K+-ATPase is the driving force behind ciliary motion, our results also suggest that in warming waters A. anatina may be more tolerant at sustaining vigorous ciliary action (associated with elevated respiration rates and filter-feeding) than U. tumidus. Overall, our results indicate great flexibility of the mussel's ecophysiological characteristics as response to changing conditions.

Submicron Plastic Adsorption by Peat, Accumulation in Sphagnum Mosses and Influence on Bacterial Communities in Peatland Ecosystems.

The smallest fraction of plastic pollution, submicron plastics (SMPs <1 µm) are expected to be ubiquitous in the environment. No information is available about SMPs in peatlands, which have a key role in sequestering carbon in terrestrial ecosystems. It is unknown how these plastic particles might behave and interact with (micro)organisms in these ecosystems. Here, we show that the chemical composition of polystyrene (PS) and poly(vinyl chloride) (PVC)-SMPs influenced their adsorption to peat. Consequently, this influenced the accumualtion of SMPs by Sphagnum moss and the composition and diversity of the microbial communities in peatland. Natural organic matter (NOM), which adsorbs from the surrounding water to the surface of SMPs, decreased the adsorption of the particles to peat and their accumulation by Sphagnum moss. However, the presence of NOM on SMPs significantly altered the bacterial community structure compared to SMPs without NOM. Our findings show that peatland ecosystems can potentially adsorb plastic particles. This can not only impact mosses themselves but also change the local microbial communities.

Ultra-acute exposure to cadmium does not impair whitefish sperm motility.

Cadmium (Cd) exposure can impair the traits of aquatic animals associated with reproduction. In natural lakes Cd is typically detected at concentrations below 0.001 mg l-1 . The authors investigated the impact of ultra-acute Cd exposure on sperm motility in European whitefish (Coregonus lavaretus). They activated sperm with water containing various nominal concentrations of Cd and recorded sperm motility parameters. Only the highest Cd concentration (500 mg l-1 ) was associated with decreased sperm swimming velocity and increases in both the percentage of static cells and curvature of the sperm swimming trajectory. The results indicate that environmentally realistic concentrations of Cd during the sperm motility activation are not critically harmful to male C. lavaretus fertilization potential.

Range expansion and reproduction of the ectoparasitic deer ked (Lipoptena cervi) in its novel host, the Arctic reindeer (Rangifer tarandus tarandus), in Finland.

The deer ked (Lipoptena cervi) is a harmful ectoparasite that emerged in the reindeer herding area of Finland in 2006. To understand the current range and the intensity of infestations on its novel reindeer host, we studied deer ked pupae collected from reindeer and moose bedding sites and conducted a questionnaire survey among the managers of 18 reindeer herding cooperatives in the southern part of the reindeer herding area. Our study confirmed that the deer ked can survive and successfully reproduce on reindeer through winter and that flying deer keds had been observed in reindeer wintering areas during several autumns in twelve cooperatives. The pupae originating from reindeer were smaller and showed lower hatching rates than the pupae from moose. The present results indicate that the range of the deer ked infestations on reindeer in Finland expanded during the recent 5 years, now reaching 14 cooperatives and bordering an area south of approximately 66° N 25° E in the west and 65° N 29° E east.

The signal crayfish (Pacifastacus leniusculus) in Lake Tahoe (USA) hosts multiple Aphanomyces species.

The genus Aphanomyces (Oomycetes) comprises approximately 50 known species of water molds in three lineages. One of the most notorious is Aphanomyces astaci, the causative agent of crayfish plague. In this study, fresh isolates of Aphanomyces were collected from 20 live specimens of the signal crayfish Pacifastacus leniusculus (Dana, 1852) from Lake Tahoe, California, providing 35 axenic cultures of A. astaci as well as two apparently undescribed Aphanomyces spp. isolates. Based on the results of ITS-, chitinase-, mitochondrial rnnS- and rnnL-sequences and microsatellite markers combined, the Lake Tahoe A. astaci isolates were identical to isolates of A. astaci B-haplogroup commonly detected in Europe, and infection experiments confirmed their high virulence towards noble crayfish. One of the two undescribed Aphanomyces spp. isolates was highly similar to an Aphanomyces lineage detected previously in crustacean zooplankton (Daphnia) in Central Europe, while the other was distinct and most closely related (ITS sequence similarity of 93%) to either A. astaci or to Aphanomyces fennicus isolated recently from Astacus astacus in Finland. Neither of the two Aphanomyces spp. isolates caused crayfish mortality under experimental conditions. Our results indicate that the populations of North American signal crayfish can act as carriers of both pathogenic and non-pathogenic Aphanomyces at the same time. Furthermore, considering that a limited number of crayfish individuals from a single location yielded multiple distinct Aphanomyces isolates, our results suggest that substantial species diversity within this genus remains undescribed.

Eco-immunology in the cold: the role of immunity in shaping the overwintering survival of ectotherms.

The effect of temperature on physiology mediates many of the challenges that ectotherms face under climate change. Ectotherm immunity is thermally sensitive and, as such, environmental change is likely to have complex effects on survival, disease resistance and transmission. The effects of temperature on immunity will be particularly profound in winter because cold and overwintering are important triggers and regulators of ectotherm immune activity. Low temperatures can both suppress and activate immune responses independent of parasites, which suggests that temperature not only affects the rate of immune responses but also provides information that allows overwintering ectotherms to balance investment in immunity and other physiological processes that underlie winter survival. Changing winter temperatures are now shifting ectotherm immunity, as well as the demand for energy conservation and protection against parasites. Whether an ectotherm can survive the winter will thus depend on whether new immune phenotypes will shift to match the conditions of the new environment, or leave ectotherms vulnerable to infection or energy depletion. Here, we synthesise patterns of overwintering immunity in ectotherms and examine how new winter conditions might affect ectotherm immunity. We then explore whether it is possible to predict the effects of changing winter conditions on ectotherm vulnerability to the direct and indirect effects of parasites.

Sperm pre-fertilization thermal environment shapes offspring phenotype and performance.

The sperm pre-fertilization environment has recently been suggested to mediate remarkable transgenerational consequences for offspring phenotype (transgenerational plasticity, TGB), but the adaptive significance of the process has remained unclear. Here, we studied the transgenerational effects of sperm pre-fertilization thermal environment in a cold-adapted salmonid, the European whitefish (Coregonus lavaretus). We used a full-factorial breeding design where the eggs of five females were fertilized with the milt of 10 males that had been pre-incubated at two different temperatures (3.5°C and 6.5°C) for 15 h prior to fertilization. Thermal manipulation did not affect sperm motility, cell size, fertilization success or embryo mortality. However, offspring that were fertilized with 6.5°C-exposed milt were smaller and had poorer swimming performance than their full-siblings that had been fertilized with the 3.5°C-exposed milt. Furthermore, the effect of milt treatment on embryo mortality varied among different females (treatment×female interaction) and male-female combinations (treatment×female×male interaction). Together, these results indicate that sperm pre-fertilization thermal environment shapes offspring phenotype and post-hatching performance and modifies both the magnitude of female (dam) effects and the compatibility of the gametes. Generally, our results suggest that short-term changes in sperm thermal conditions may have negative impact for offspring fitness. Thus, sperm thermal environment may have an important role in determining the adaptation potential of organisms to climate change. Detailed mechanism(s) behind our findings require further attention.

MtDNA allows the sensitive detection and haplotyping of the crayfish plague disease agent Aphanomyces astaci showing clues about its origin and migration.

The oomycete Aphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogen A. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studied A. astaci strains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating from Orconectes sp., Pacifastacus leniusculus and Astacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from the P. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating from Procambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from the Orconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of the A. astaci haplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.

Metabolic rate associates with, but does not generate covariation between, behaviours in western stutter-trilling crickets, Gryllus integer.

The causes and consequences of among-individual variation and covariation in behaviours are of substantial interest to behavioural ecology, but the proximate mechanisms underpinning this (co)variation are still unclear. Previous research suggests metabolic rate as a potential proximate mechanism to explain behavioural covariation. We measured the resting metabolic rate (RMR), boldness and exploration in western stutter-trilling crickets, Gryllus integer, selected differentially for short and fast development over two generations. After applying mixed-effects models to reveal the sign of the covariation, we applied structural equation models to an individual-level covariance matrix to examine whether the RMR generates covariation between the measured behaviours. All traits showed among-individual variation and covariation: RMR and boldness were positively correlated, RMR and exploration were negatively correlated, and boldness and exploration were negatively correlated. However, the RMR was not a causal factor generating covariation between boldness and exploration. Instead, the covariation between all three traits was explained by another, unmeasured mechanism. The selection lines differed from each other in all measured traits and significantly affected the covariance matrix structure between the traits, suggesting that there is a genetic component in the trait integration. Our results emphasize that interpretations made solely from the correlation matrix might be misleading.

Summer time predation on the obligatory off-host stage of an invasive ectoparasite.

Predation can regulate populations and strongly affect invasion success of novel prey. The deer ked (Lipoptena cervi; Linnaeus 1758) is an invasive ectoparasite of cervids that spends a long period of its life cycle outside the host. Prior to this study, virtually nothing was known about natural summer time predation on the deer ked. We aimed to evaluate the magnitude of summer time predation on L. cervi pupae in different habitats and to identify potential predators. We conducted a set of field experiments, where we exposed L. cervi pupae to various ground-dwelling vertebrate and invertebrate predators. The loss of pupae was monitored for different predator guilds. Three habitats of the moose, the main host species, were studied: (1) moist heath forest; (2) dry, logged heath forest; and (3) moist meadow. The results indicate notable summer time predation on L. cervi pupae, and the pupal predation varied within and between habitats, being lowest in the meadow habitat. We found a positive correlation between pupal loss and abundance of the common lizard (Zootoca vivipara), harvestmen (Opiliones), ground spiders (Gnaphosidae) and Formicinae-ants. We conclude that summer time predation during the pupal phase can have a notable local importance for the L. cervi abundance.

Early-life temperature modifies adult encapsulation response in an invasive ectoparasite.

Immunity of parasites has been studied amazingly little, in spite of the fact that parasitic organisms, especially the arthropod parasites, need immunity to survive their own infections to successfully complete life cycles. Long-term effects of challenging environmental temperatures on immunity have remained unstudied in insects and parasites. Our study species, the deer ked (Lipoptena cervi; Linnaeus 1758), is an invasive, blood-feeding parasitic fly of cervids. Here, it was studied whether thermal stress during the pupal diapause stage could modify adult immunity (encapsulation capacity) in L. cervi. The effect of either a low temperature or high temperature peak, experienced during winter dormancy, on encapsulation response of active adult was tested. It was found that low temperature exposure during diapause, as long as the temperature is not too harsh, had a favourable effect on adult immunity. An abnormal, high temperature peak during pupal winter diapause significantly deteriorated the encapsulation capacity of emerged adults. The frequency and intensity of extreme weather events such as high temperature fluctuations are likely to increase with climate change. Thus, the climate change might have previously unknown influence on host-ectoparasite interactions, by affecting ectoparasite's immune defence and survival.

Infection ecology of Philometra ovata (Nematoda: Philometridae) in a wild European minnow (Phoxinus phoxinus) population in Finland.

Seasonal life cycle of body cavity dwelling (BCD) Philometra ovata (Nematoda: Philometridae) has been reported in southern and central European countries, but its swim bladder dwelling (SBD) stage and northern populations have remained unstudied. In this study, we investigated the seasonal life cycle and infection ecology of P. ovata in both swim bladder and body cavity in the European minnow (Phoxinus phoxinus) in Finland. The larval P. ovata infected the swim bladder of minnows mainly in August. Female SBD P. ovata emigrated to body cavity mostly in September, grew to their full size by the end of the next June, and evacuated from minnows in July. In addition, female SBD P. ovata retarding their development and staying in swim bladder were found commonly in minnows, thus the mean monthly prevalence (6·7 ± 3·9%) and mean intensity (1·4 ± 0·8) of BCD P. ovata was lower than that of SBD P. ovata (37·8 ± 15·1% and 2·0 ± 1·5, respectively). Finally, despite the large size of BCD individuals, infection of P. ovata did not impair body condition and relative gonad size of minnows, but increased the mortality and caused physical damages in their hosts during the evacuation period.

Passive sinking into the snow as possible survival strategy during the off-host stage in an insect ectoparasite.

Abiotic and biotic factors determine success or failure of individual organisms, populations and species. The early life stages are often the most vulnerable to heavy mortality due to environmental conditions. The deer ked (Lipoptena cervi Linnaeus, 1758) is an invasive insect ectoparasite of cervids that spends an important period of the life cycle outside host as immobile pupa. During winter, dark-coloured pupae drop off the host onto the snow, where they are exposed to environmental temperature variation and predation as long as the new snowfall provides shelter against these mortality factors. The other possible option is to passively sink into the snow, which is aided by morphology of pupae. Here, we experimentally studied passive snow sinking capacity of pupae of L. cervi. We show that pupae have a notable passive snow sinking capacity, which is the most likely explained by pupal morphology enabling solar energy absorption and pupal weight. The present results can be used when planning future studies and when evaluating possible predation risk and overall survival of this invasive ectoparasite species in changing environmental conditions.

Dose-dependent mortality of the noble crayfish (Astacus astacus) to different strains of the crayfish plague (Aphanomyces astaci).

Several reports of the European crayfish species carrying a latent infection of the crayfish plague (Aphanomyces astaci) have emerged and the discussion has focused especially on the lowered virulence of As-genotypes behind decreased mortality. The aim of this study was to compare the killing rate of different A. astaci strains in controlled infection experiments. Two separate infection experiments with three A. astaci strains (UEFT2B (As), Evira6462/06 (As) and UEF8866-2 (PsI)) were made to compare the noble crayfish populations from the Lake Viitajärvi, Tervo, (Expt I) and the Lake Mikitänjärvi, Hyrynsalmi (Expt II). In the Expt III, the Lake Koivujärvi population noble crayfish were infected with A. astaci strains UEF8866-2 (PsI) and Evira6462/06 (As) using different dosages (1, 10, 100 and 1000sporesml(-1)) of A. astaci zoospores. The results confirmed that PsI-genotype strain is highly virulent and kills all the crayfish within a few days. The tested two As-genotype strains caused the mortalities more slowly, and part of the challenged crayfish survived until the end of the follow-up period. Our results also confirmed the variance of virulence among A. astaci strains within the As-genotype and demonstrated that the mortality is dependent on the number of zoospores used in the infections. It also appeared, that some noble crayfish populations show increased resistance towards the crayfish plague, especially against the As-genotype of A. astaci.

Acute impacts of the deer ked (Lipoptena cervi) infestation on reindeer (Rangifer tarandus tarandus) behaviour.

Blood-sucking ectoparasites have often a strong impact on the behaviour of their hosts. The annual insect harassment of reindeer (Rangifer tarandus tarandus) has increased in the southern part of the Finnish reindeer herding area because of the recent invasion of a blood-feeding ectoparasitic louse-fly, the deer ked (Lipoptena cervi). We studied the impact of the deer ked on the behaviour of reindeer. Twelve reindeer were infested with a total of 300 keds/reindeer on six occasions in a 5-week period during the deer ked flight season in autumn, while six non-infested reindeer were used as controls. Behavioural patterns indicating potential stress were monitored by visual observation from August to December. The infested reindeer displayed more incidences of restless behaviour than the controls. Shaking and scratching were the most common forms of restless behaviour after infestation of deer keds. Increased grooming was also observed after the transplantation and also later, 1 month after the infestation. Based on the results, the deer ked infestation can cause acute behavioural disturbance in reindeer and, thus, could pose a potential threat to reindeer welfare. Antiparasitic treatment with, e.g. ivermectin, may increase the welfare of parasitized reindeer by reducing deer keds. If the deer ked infestation intensity on the reindeer herding area increases and restless behaviour of reindeer becomes more common, the present results can help in further evaluation of the duration and magnitude of behavioural changes.

Selection among critically endangered landlocked salmon (Salmo salar m. sebago) families in survival and growth traits across early life stages and in different environments.

Endangered wild fish populations are commonly supported by hatchery propagation. However, hatchery-reared fish experience very different selective pressures compared to their wild counterparts, potentially causing genotype-by-environment interactions (G × E) in essential fitness traits. We experimentally studied early selection in a critically endangered landlocked Atlantic salmon population, first from fertilization to the swim-up stage in a common hatchery setting, and thereafter until the age of 5 months in two contrasting rearing environments. Swim-up progeny were moved either to standard indoor hatchery tanks involving conventional husbandry or to seminatural outdoor channels providing only natural food. After the first summer, sampled survivors were assigned to their families by genotyping. Early survival until the swim-up stage was mostly determined by maternal effects, but also involved significant variation due to sires and full-sib families (potential genetic effects). High on-growing survival in hatchery tanks (88.7%) maintained a more even distribution among families (relative share 1.5%-4.2%) than the seminatural environment (0.0%-5.4%). This heterogeneity was mostly maternal, whereas no independent paternal effect occurred. Heritability estimates were high for body size traits in both environments (0.62-0.69). Genetic correlations between the environments were significantly positive for body size traits (0.67-0.69), and high body condition in hatchery was also genetically linked to rapid growth in the seminatural environment (0.54). Additive and phenotypic growth variation increased in the seminatural environment, but scaling effects probably played a less significant role for G × E, compared to re-ranking of genotypes. Our results suggest that not only maternal effects, but also genetic effects, direct selection according to the environmental conditions experienced. Consistently high genetic variation in growth implies that, despite its low overall genetic diversity and long history in captive rearing (>50 years), this landlocked Atlantic salmon population still possesses adaptive potential for response to change from hatchery rearing back to more natural conditions.

Female ornaments revisited - are they correlated with offspring quality?

The evolution and signalling content of female ornamentation has remained an enduring challenge to evolutionary biologists, despite the fact that secondary sexual characters are widespread in females. While females usually invest significant amounts of their resources, including carotenoids, in offspring, all the resources allocated to elaborate ornamentation reduce resources available for other purposes. This may in turn constrain female fitness leading to dishonest female signalling. We review the literature for empirical studies on mutually ornamented species with conventional sex roles, by focusing on the association between female ornaments and quality of their offspring. We found 43 papers where 33 (77%) are bird-studies, nine (21%) are on fishes, and one (2%) is a lizard-study. Nine of these report negative, 14 non-existing, and 20 positive associations between female ornament and offspring quality. Eighteen of the bird studies (55%) show a positive association between the two traits investigated, whereas five (15%) of the studies report a negative association. The number of fish studies, although few, is skewed in the opposite direction with two (22%) and four (44%) studies supporting positive and negative association, respectively. A minority of studies on carotenoids-based ornaments reports a positive association (4 of 18 studies, or 22%) between the traits, which is low compared to studies on non-carotenoids-based ornaments (16 of 25 studies, or 64%). The above-mentioned relative large number of studies with negative association, especially common in studies on fishes and in carotenoids-based-ornaments, challenges the generality of the direct selection hypothesis to account for female fineries. This is important because this hypothesis seems to have strong support in recent literature on the topic. In the present paper, we also propose possible explanations for the observed differences between taxa and suggest directions and ideas for future research on the evolution of female ornamentation.

Dominance is not always an honest signal of male quality, but females may be able to detect the dishonesty.

Females prefer dominant males as mating partners in numerous species. Male dominance rank is considered as an honest signal of male quality, because only healthy males in good condition are thought to be able to win fights with other males. Here, we tested whether activation of the immune system influences the success of males in male-male competition and mating in the field cricket, Gryllus integer. We activated the immune system of males with a nylon monofilament (to mimic a parasitoid larva), and arranged fights between male pairs to assess male dominance and associated mating success. Activation of the immune system with nylon monofilament substantially enhanced the fighting success of males during male-male competition but had no effect on mating success. However, sham-manipulation (a wound only) did not have any effect on fighting success although females mated more often with dominant males. Our study suggests that when male crickets meet an apparent survival threat they may behave more dominantly, probably owing to terminal investment. Male success during male-male competition is not always an honest signal of males' quality, but females may be able to detect this dishonesty.

Crayfish population size under different routes of pathogen transmission.

We present an epidemiological model for the crayfish plague, a disease caused by an invasive oomycete Aphanomyces astaci, and its general susceptible freshwater crayfish host. The pathogen shows high virulence with resulting high mortality rates in freshwater crayfishes native to Europe, Asia, Australia, and South America. The crayfish plague occurrence shows complicated dynamics due to the several types of possible infection routes, which include cannibalism and necrophagy. We explore this complexity by addressing the roles of host cannibalism and the multiple routes of transmission through (1) environment, (2) contact, (3) cannibalism, and (4) scavenging of infected carcasses. We describe a compartment model having six classes of crayfish and a pool of crayfish plague spores from a single nonevolving strain. We show that environmental transmission is the decisive factor in the development of epidemics. Compared with a pathogen-free crayfish population, the presence of the pathogen with a low environmental transmission rate, regardless of the contact transmission rate, decreases the crayfish population size with a low risk of extinction. Conversely, a high transmission rate could drive both the crayfish and pathogen populations to extinction. High contact transmission rate with a low but nonzero environmental transmission rate can have mixed outcomes from extinction to large healthy population, depending on the initial values. Scavenging and cannibalism have a relevant role only when the environmental transmission rate is low, but scavenging can destabilize the system by transmitting the pathogen from a dead to a susceptible host. To the contrary, cannibalism stabilizes the dynamics by decreasing the proportion of infected population. Our model provides a simple tool for further analysis of complex host parasite dynamics and for the general understanding of crayfish disease dynamics in the wild.

Exposure protocol for ecotoxicity testing of microplastics and nanoplastics.

Despite the increasing concern about the harmful effects of micro- and nanoplastics (MNPs), there are no harmonized guidelines or protocols yet available for MNP ecotoxicity testing. Current ecotoxicity studies often use commercial spherical particles as models for MNPs, but in nature, MNPs occur in variable shapes, sizes and chemical compositions. Moreover, protocols developed for chemicals that dissolve or form stable dispersions are currently used for assessing the ecotoxicity of MNPs. Plastic particles, however, do not dissolve and also show dynamic behavior in the exposure medium, depending on, for example, MNP physicochemical properties and the medium's conditions such as pH and ionic strength. Here we describe an exposure protocol that considers the particle-specific properties of MNPs and their dynamic behavior in exposure systems. Procedure 1 describes the top-down production of more realistic MNPs as representative of MNPs in nature and particle characterization (e.g., using thermal extraction desorption-gas chromatography/mass spectrometry). Then, we describe exposure system development for short- and long-term toxicity tests for soil (Procedure 2) and aquatic (Procedure 3) organisms. Procedures 2 and 3 explain how to modify existing ecotoxicity guidelines for chemicals to target testing MNPs in selected exposure systems. We show some examples that were used to develop the protocol to test, for example, MNP toxicity in marine rotifers, freshwater mussels, daphnids and earthworms. The present protocol takes between 24 h and 2 months, depending on the test of interest and can be applied by students, academics, environmental risk assessors and industries.