Plastic waste interferes with chemical communication in aquatic ecosystems

Environmental pollution with plastic waste has gained increasing attention, as the contamination of aquatic habitats poses a challenge to these ecosystems. Plastic waste has direct negative effects on animals such as reduced growth rate, fecundity or life span. However, the indirect effects of plastic waste, which has the ability to sorb chemicals from the surrounding media, on chemical communication have yet to be investigated. Chemical communication is crucial for aquatic organisms, e.g., to avoid predation. The planktonic water flea Daphnia (Crustacea), an important link between trophic levels, relies on info-chemicals (kairomones) to assess its current predation risk and to form inducible defences. We show that plastic waste, composed of high-density polyethylene (HDPE) and polyethylene terephthalate (PET) interferes with the formation of inducible defences in Daphnia longicephala when exposed to a combination of kairomones of Notonecta glauca and plastic waste. D. longicephala shows a reduction in all defensive traits, including body length, crest width and time until primiparity, compared to exposure to solely kairomone conditioned media. Plastic waste in the absence of kairomones had no effect on defensive traits. Since it is vital to adjust these defences to the current predation risk, any misperception can have far-reaching ecological consequences. Therefore, plastic waste can have indirect effects on organisms, which may manifest at the community level.

Kairomone characterization: Kairomones are not chemically defined compounds but they have an ecological definition: Kairomones are semiochemicals that bring benefit to the acceptor of the chemical signal while not being beneficial for the emitter. However, the nature of those chemicals is not known in most cases, since activity might be a result of several chemicals that act synergistically (see for example Pohnert et al 25 . So far the following characteristics have been described for kairomones of several predators: -low molecular weight (˂500 Da) 21,22 -non-volatile, and anionic compound of medium polarity 21,22 -extreme pH (0.8\pH\14.0) and temperature (-20°C-120°C) stability -proteinase and peptidase resistant 21,22 -hydroxyl groups are revealed as essential for biological activity of the kairomone 17,22 -methyl groups and amine groups might be a substantial part of kairomones -loses its activity under non-sterile conditions due to microbial degradation 21 .
As the exact chemical structure of kairomones is not known in many cases, a direct measurement of kairomone concentration is not feasible. Since the expression of the morphological traits is following a dose-response curve, kairomone concentration is measured via the reaction norm of the phenotypically plastic traits.

Supplementary Discussion
Effects of leached additives An increase in crest width was found in the C+HDPE treatment, which is absent of kairomones.
Additives which may leach out of the plastics may evoke those effects. Yang et al. S1 were able to show that different plastic products used for food packaging leached additives, which may have endocrine activity, in saline medium as well as in ethanol. Among those products were some made of the polymers HDPE and PET, both polymers found in our experimental setup. Of the two polymers HDPE only leached detectable additives in 61% of scenarios whereas PET leached in 100%. Yet, this leaching was measured after the polymers were exposed to ultraviolet radiation (wavelength 254nm) and incubated at a temperature of 37° C for a duration of 72h. As leaching is enhanced by UV radiation, as well as high temperature it is unlikely that leaching took place in our experimental, as there was no UV exposure, temperature was 17°C lower and experimental time was a third of the time used in the study by Yang et al. 41 . Furthermore, only a single morphological parameter is affected in our study. If leachates would stimulate the development of defensive structures, it is anticipated that every morphological parameter would be affected. In addition, as leaching of additives from PET was more distinct in the study of Yang et al. S1 , we would have expected morphological changes rather in den C+PET daphnids compared to the C+HDPE daphnids were a slight crest induction was observed. Since the effect of additives was not the focus of our study we did not conduct any leachate analysis of the plastics and the surrounding medium. Hence, the discussion on the effects of possible leachates is highly speculative. However, that possible leachates from the used plastics did not affect Daphnia in our study is further supported, by the study of Lithner et al. S2 , where the effects on D. magna by leachates from various plastics were tested. Here 20 g of plastic were dispersed in 200 ml of deionized water and shaken for 24 h at a temperature of 20 ± 2°C, making conditions similar to our experiment. Yet, the leachates of HDPE did not show any effects on Daphnia.
Value of our study for future studies on inducible defenses For our positive control, namely, the glass treatment, the following insights can be applied to past and future studies investigating kairomones. In our experiment, the I+GLASS daphnids were smaller in body length (BL) and crest width (CW) and reached primiparity earlier than the solely kairomone-exposed daphnids (I). These findings imply that glass also adsorbs kairomones to its surface, thereby reducing the perceivable kairomone concentration. Usually, authors state the ratio of predators per liter of medium in their induction experiments. With our results in mind, we suggest that the surface-to-volume ratio of the experimental container should also be reported to facilitate comparability among studies.

Fig. S 3: Effects of kairomone on morphological and life history parameters of D. longicephala: A)
body length of daphnids exposed to kairomones compared to control daphnids lacking exposure to kairomones; B) crest width of daphnids exposed to kairomones compared to control daphnids lacking exposure to kairomones; C) duration in days until daphnids reach primiparity when exposed to kairomones compared to control daphnids lacking exposure to kairomones. Error bars indicate standard error of means (±1SE) Asterisks represent level of significance when treatments are compared with induction (I) or control (C) treatments: n.s. = p ≥ 0.05; * = p ≤ 0.05; *** = p ≤ 0.001