Invasive species represent a significant threat to native biodiversity. The Himalayan Balsam Impatiens glandulifera is an annual plant, which is invasive in Europe and often inhabits the riparian zone. It produces several secondary metabolites causing, for example, growth inhibition of terrestrial plants and invertebrates. One of these metabolites is the quinone 2-methoxy-1,4-naphthoquinone (2-MNQ). The compound gets washed out from the above-ground parts of the plant during precipitation and may then leach into nearby waterbodies. Despite some evidence for the allelopathic effect of plant secondary metabolites on terrestrial invertebrates, little is known about how 2-MNQ affects the survival or development of aquatic dipteran larvae, despite the importance of this functional group in European freshwaters. Here, we investigated the effects of 2-MNQ on larvae of the river keystone species Chironomus riparius in acute and chronic scenarios. The toxicity of 2-MNQ towards the first and the fourth larval stage was determined in a 48-hour acute exposure assay. We show that 2-MNQ has a negative impact on the development, growth and survival of C. riparius. The LC₅₀ of 2-MNQ was 3.19 mg/l for the first instar and 2.09 mg/l for the fourth instar. A ten-day chronic exposure experiment, where the water was spiked with 2-MNQ, revealed that 2-MNQ had a significantly negative impact on larval body size, head capsule size, body weight, development and survival. These results demonstrate the negative impact of the secondary metabolite 2-MNQ from the terrestrial plant I. glandulifera on a crucial macroinvertebrate inhabiting the adjacent stream ecosystem in riverine ecosystems. This may lead to a decline in population size, resulting in cascading effects on the food web.

Allelopathy, benthic macroinvertebrates, ecotoxicity, invasive species, 2-methoxy-1, 4-naphthoquinone

The riparian zone, the transition zone between terrestrial and freshwater ecosystems, is amongst the most diverse habitats worldwide. The vegetational and structural diversity acts as a refuge for small mammals hiding in shrubs, trees serve as perching and nesting sites for birds and fallen wood debris provides resources for terrestrial as well as aquatic invertebrates (Naiman and Décamps 1997). Hence, it supplies the freshwater system with allochthonous organic and inorganic materials (Gregory et al. 1991; Naiman et al. 1993). A major threat to the riparian zone, the adjacent freshwater ecosystems and their biodiversity are invasive alien species (Pyšek 1994). In times of globalisation, the frequency of biological invasions is rising continuously in every type of habitat and taxonomic group (Mills et al. 1993). Species are frequently introduced through the freight or ballast tanks of ships, planes and trucks, whose traffic have risen strongly because of increasing trade (Verling et al. 2005; Hulme 2009).

Invasive plants can impair native species by producing allelopathic metabolites. The Japanese knotweed Fallopia japonica, for example, produces resveratrol, amongst other chemicals, which has been found to have inhibitory effects on seed germination and seedling growth of various plant species, potentially influencing the structure and composition of plant communities in invaded areas (Abgrall et al. 2018). Rhododendron ponticum, another invasive plant species, is known to significantly impact aquatic habitats through multifaceted ecological interactions (Erfmeier and Bruelheide 2010). The colonisation of freshwater ecosystems by R. ponticum leads to alterations in water quality, light availability and nutrient cycling (Vitousek 1990; Urgenson and Reichard 2007). The shading effect induced by its dense canopy significantly impacts algal growth, while the release of leachates from its leaves influences microbial and fungal communities (Hladyz et al. 2011; Monk et al. 2014; Jones et al. 2019). Furthermore, the slower decomposition rates of R. ponticum litter compared to native plants in waterbodies contributes to organic matter accumulation (Jones et al. 2019). Studies on leachates from Senecio jacobaea or Petasites hybridus have demonstrated notable concentrations of phytotoxins, like pyrrolizidine alkaloids (PA), originating from these plants in small streams and seepage water (Kisielius et al. 2020). Additionally, precipitation amplified PA concentrations by a factor of ten in stream water, posing potential challenges for aquatic ecosystems, particularly during the rainy season (Kisielius et al. 2020).

Another well-known example of an invasive alien species in riparian habitats is the Himalayan Balsam Impatiens glandulifera. It belongs to the family of the Balsaminaceae, reaches a height of up to 2.5 m, can disperse up to 2500 seeds per mature plant in a radius of 10 m and achieves up to 90% cover of invaded plots (Beerling and Perrins 1993; Hejda et al. 2009; Chapman and Gray 2012). The pathways of introduction typically include trade with the plant and seed mixture contamination (Millane and Caffrey 2014). Dispersal can also happen through wildlife or waterways, as the seeds are adapted for water dispersal (Pysek and Prach 1995). A reason for its invasive success is the release of allelopathic secondary metabolites like the quinone 2-methoxy-1,4-naphthoquinone (2-MNQ) (Chapelle 1974; Ruckli et al. 2014a; Meyer et al. 2021). 2-MNQ is released from the roots of I. glandulifera into the ground (Lobstein et al. 2001; Ruckli et al. 2014a). As the substance leaches into the ground, it inhibits the growth of seedlings and juveniles of native co-occurring plants, like the stinging nettle Urtica dioica or inhibits the arbuscular mycorrhiza colonisation of sycamore saplings (Ruckli et al. 2014a, b; Bieberich et al. 2018). 2-MNQ is further washed off the leaves during precipitation leading to a pulsed introduction of this allelochemical in high concentrations into adjacent habitats, including waterbodies in riparian habitats (Lobstein et al. 2001; Ruckli et al. 2014a). Run-off of I. glandulifera has been shown to inhibit the growth of the aquatic green algae Acutodesmus obliquus and also affects the mortality, the growth and the reproduction of Daphnia magna, a key species in standing freshwater habitats, building the link between primary producers and higher trophic levels (Brett and Goldman 1997; Diller et al. 2022). However, it is not known yet if 2-MNQ of the invasive alien species I. glandulifera has an impact on riverine arthropods and ecosystems.

Amongst running waters, rivers belong to the most diverse ecosystems, providing the potential for various ecological niches due to the richness of different and heterogeneous habitat patches (Lake 2000). Here, benthic macroinvertebrates inhabit almost every ecological niche and act as links between the input of allochthonous material and higher trophic levels such as fish (Richardson 1993). Chironomidae (non-biting midges) are essential members of the benthic macroinvertebrate fauna in riverine ecosystems, as they frequently represent the most abundant species group (Armitage et al. 1995). They are often used as bioindicators for water quality and play a significant role in assessing the ecological state and health of flowing waters (Hellawell 1986) due to their high susceptibility to anthropogenic pollutants, such as heavy metals (de Bisthoven et al. 1992), pesticides (Tassou and Schulz 2009) or antibiotics (Park and Kwak 2018). In contrast to these pollutants, the effects of 2-MNQ released by I. glandulifera have as yet not been tested on this key species of running waters.

This paper, therefore, aimed to examine the effects of the allelopathic secondary metabolite 2-MNQ on the growth, development and survival of Chironomus riparius. We performed acute immobilisation tests, as well as low-dose chronic exposure experiments using concentrations that are comparable to those released during rain events in nature (Ruckli et al. 2014a).

Our results show that 2-MNQ has the potential to impair the survival and development of C. riparius after acute 48 hour and chronic 10-day exposure. We determined the LC₅₀ after 48 h for the first instar larvae of C. riparius at a 2-MNQ concentration of 3.16 mg/l and 2.09 mg/l for the fourth instar larvae. Larvae of C. riparius exposed to a concentration of 2 and 3 mg/l 2-MNQ in the 10-day chronic exposure experiment had significantly increased mortality, reduced body- and head capsule size, as well as reduced body weight. They were further delayed in their development and showed a significantly higher proportion of individuals with deformed and abnormal head capsules.

The doses applied in the acute (max. 8 mg/l) and chronic (max. 3 mg/l) toxicity tests were below the concentration reported to be leached from one single plant after rain events (12.21 mg/l) (Ruckli et al. 2014a). I. glandulifera is known to grow densely and crowd out other plant species by forming monocultures along riverbanks (Pattison et al. 2016; Čuda et al. 2017). Consequently, it could be assumed that rain events and subsequent run-off have a substantial impact on the survival and development of freshwater invertebrates when an I. glandulifera monoculture surrounds the waterbody. This of course depends on the velocity of the river and the water volume of the waterbody, which are both important factors in terms of the dilution effects of xenobiotics, where a lower dilution increases the bioaccumulation and contamination risk (Keller et al. 2014; Dris et al. 2015). As a result, benthic macroinvertebrates living in small and slowly running waters should be more susceptible to incoming 2-MNQ because of a higher accumulation risk (Logan and Brooker 1983; Clements 1994).

It has already been shown that low concentrations of 1.5 mg/l 2-MNQ can significantly impair the growth and survival of individuals of the freshwater key species Daphnia magna (Diller et al. 2023). In comparison, the closely-related compound plumbagin (2-methyl-5-hydroxy-1,4-naphthoquinone) from the roots of Plumbago zeylanica shows toxic effects on survival at 1 mg/l towards marine copepods and the synthetic derivate of 2-MNQ, menadione (2-methyl-1,4-naphthoquinone) has an LC₅₀ of 2.3 mg/l against adults of Dreissena polymorpha (Sugie et al. 1998; Wright et al. 2006). These results concerning LC values and survival analyses are in concordance with the LC₅₀ we found (2.09–3.19 mg/l) for 2-MNQ and suggest similar toxicity of 1,4-naphthoquinones towards invertebrate organisms. Responsible for the high toxicity of 2-MNQ towards invertebrates could be the high reactivity of quinones, due to electron-withdrawing carbonyl groups and redox properties, with an even higher reactivity of 1,4-naphthoquinones in an aqueous medium (Pereyra et al. 2019). This is due to a nucleophilic substitution and the interaction of non-polar and hydrophobic regions of reactants, causing irreparable damage to DNA by alkylating nucleophilic sites (Tandon and Maurya 2009; Pereyra et al. 2019).

The requirement of sediment of fourth instar larvae could be a reason for the higher toxicity of 2-MNQ, compared to the first instar. Naphthalene, for example, a structurally related compound to 2-MNQ, is known to be easily oxidised and interact with a SiO₂/air interface (Barbas et al. 1993). This can lead to a higher concentration of 2-MNQ in the sediment than in the water column, resulting in a higher exposure risk (Corpus-Mendoza et al. 2022) as sediment is crucial for the second to the fourth instar larvae of C. riparius. The sediment is required for building tubes out of silk from the salivary glands, used for nutrient acquisition and protection by the larvae (Armitage et al. 1995). However, it has to be further investigated if 2-MNQ is interacting with the SiO₂ surface of quartz sand in an aqueous environment and if that interaction increases or decreases the toxicity of 2-MNQ. Another possible explanation for the higher toxicity of 2-MNQ towards the fourth instar larvae could be that it is the last developmental stage before pupation. This could lead to higher susceptibility towards endocrine-disrupting substances like 2-MNQ, as the last larval stage of homometabolic insects requires the highest titre of ecdysteroids, to shift the larval genome towards pupal pattern formation (Smith 1985; Mitchell et al. 1999; Mitchell et al. 2007). The development of the larvae could further be impaired by 2-MNQ disrupting the function of the cytochrome P450-dependent steroid hydroxylase ecdysone-20-mono-oxygenase, which hydroxylates the inactive ecdysone to the active moulting hormone ecdysterone, which can lead to delayed moulting or in general impaired postembryonic development and inhibition of pupal formation (Smith et al. 1979; Smith 1985; Mitchell et al. 2007). Other 1,4-naphthoquinones seem to have similar effects on insects. Juglone, plumbagin, menadione and lawsone also show toxic effects on the larvae of the saturniid moth Actias lunas, evident by increased mortality and developmental time (Thiboldeaux et al. 1994). Another possible explanation for why 2-MNQ interferes with moulting is that it could inhibit the chitin synthetase of insect larvae, which is crucial for the moulting process, as shown for the naturally-occurring plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) originating from Plumbago capensis towards the larvae of Bombyx mori (Kubo et al. 1983). The darker head capsule may be explained by 1,4-naphthoquinones’ ability to bind to and modify the colour of chitosan (Muzzarelli et al. 2003). This could also be the case for chitin, the acetylated version of chitosan (Dutta et al. 2004).

Even though some chironomid species are known for their extreme tolerance towards environmental conditions like pH, temperature, oxygen content and even salinity, they are susceptible to anthropogenically induced pollution, drugs and other endocrine-disrupting substances (Vermeulen et al. 2000; Taenzler et al. 2007; Serra et al. 2017). If their biomass is significantly reduced, there could be a severe impact on higher trophic levels, depending on the chironomids as a food source. This could be shown in modelled exposure scenarios of the Chinook salmon (Oncorhynchus tshawytscha) and the associated macroinvertebrate prey community, as some pesticides only affected the growth rates of salmon populations by reducing the availability of prey (Macneale et al. 2014). In addition, also terrestrial predators like bats and birds are highly dependent on emerging chironomids as food sources, leading to a potential food deficiency or at least increased energy demands due to an increased predation radius and time away from the nest when breeding in those organisms (Barclay 1991; Martin et al, 2000; Jackson et al. 2020).

For the assessment of the impact of 2-MNQ on riverine ecosystems, it might be essential to investigate the potentially different sensitivity of various macroinvertebrates, as C. riparius is known to display a comparatively greater tolerance towards deteriorating water quality (Pinder 1986; Jiang et al. 2021; Leitner et al. 2021). Understanding these interspecific differences in sensitivity may be crucial for risk assessment and will, therefore, serve as a basis for effective conservation and management strategies.

This study reveals substantial acute and chronic toxicity of 2-MNQ towards the larvae of C. riparius. Individuals exposed to concentrations of 2 mg/l upwards showed a significantly reduced body size and head capsule size, a significantly reduced dry weight per larvae, developmental abnormalities and increased mortality compared to unexposed individuals. I. glandulifera is spreading extensively around the world, building monocultures across riverine ecotones and even invading forest ecosystems. The exposure risk to 2-MNQ could be highly increased when larger areas are covered by the plants at high densities along riverbanks. This can result in higher amounts of 2-MNQ leaching into aquatic ecosystems after precipitation, ultimately increasing its concentration within the waterbody. Our findings underscore the critical need for monitoring this neophyte, emphasising the imperative to focus on controlling its spread. This attention is vital to safeguard ecosystem functions of flowing waters.

Future research should include how riverine communities adapt to and are influenced by allelopathic substances, addressing also species interactions and resilience of these ecosystems.