This study assessed how DOC affects the hatching patterns of resting eggs of a common tropical cladoceran, Diaphanosoma birgei. We observed that the joint exposure of resting eggs to chemical and physical effects of DOC (treatments A, B and D) did not affect the hatchling numbers, but affected the time to first hatching in opposing directions depending on DOC concentration, i.e. 50 mg/l DOC decreased the time to first hatching, while 100 mg/l DOC increased it. Similarly, we observed that physical effects of DOC affected hatchling numbers and time to hatching in opposing directions (treatments A, C and E), since treatments with 50 mg/l DOC displayed higher hatchling numbers and lower time to hatching, while the opposite pattern was observed in 100 mg/l treatments. Because comparisons of treatments with the same DOC concentration (treatments B x C, and D x E) did not differ in any response variable, we conclude that chemical effects of DOC have weak to no effects on hatching patterns. As such, physical effects of DOC, especially through light attenuation under increasing DOC concentrations, seems to be the main cause of DOC changing hatching patterns of resting eggs. These findings partially corroborate our hypothesis that increasing DOC reduces hatchling numbers and increases the time to hatching, because responses of resting eggs to increasing DOC were non-linear and responded solely to light attenuation.
Light attenuation by DOC is mainly caused by the ability of humic substance carbon chains to reflect light (Danilov and Ekelund 2001; Huovinen et al. 2003; Faithfull et al. 2015). Light and temperature are largely acknowledged as the main triggers for hatching zooplankton resting eggs (Vandekerkhove et al., 2005). The light effect on hatching was first acknowledged in temperate systems, where abrupt changes in the photoperiod over seasons signalize changes in food abundance and other environmental conditions (Taghavi et al. 2013; Stewart et al. 2017). The photoperiod in tropical ecosystems where the resting eggs of this study originated does not drastically change over seasons, but light intensity may still vary as water depth and turbidity changes, thus potentially influencing hatching patterns. More recently, light intensity has been pointed out as an additional important factor for terminating diapause, as Daphnia magna resting eggs exposed to the same photoperiod under different light intensities showed higher hatching rates under higher light intensities (Slusarczyk and Flis, 2019). Our results are thus in agreement with earlier studies showing that light affects hatching patterns, but contrast with the results from Slusarczyk and Flis (2019) as intermediate light intensities promoted higher hatching rates in our experiment. Thus, we suggest for the first time that DOC may affect hatching patterns of resting eggs by attenuating light intensity.
Contrary to our expectations, DOC had no effects on hatching patterns when in direct contact with resting eggs. DOC is acknowledged to directly affect several life history parameters of cladocerans, as HS may be taken up by organisms and interact with biochemical constituents and signaling pathways (Steinberg et al., 2006; Suhett et al., 2011; Nova et al., 2018). Additionally, DOC may dye the body surface of cladocerans turning them darker. If DOC also dyes the external layer of resting eggs, then the amount of light reaching resting eggs will diminish and hatching patterns may change. It is possible that the exposure time of resting eggs to chemical effects of DOC in our experiment was not long enough to allow DOC to dye the surface of resting eggs. However, this could be tested by exposing resting eggs to DOC under inhibiting conditions for hatching for a longer time, and then testing for changes in surface pigmentation and hatching patterns of resting eggs.
The absence of chemical DOC effects on hatching patterns may be a consequence of the ability of resting eggs to tolerate high concentrations of chemical stressors, mainly due to the resistance and low permeability of resting eggs’ superficial layers. Indeed, earlier studies testing the effects of salinity, heavy metals and organic compounds other than DOC have shown that resting eggs can resist the chemical stress (Yan et al., 2004; Jiang et al., 2007; Aránguiz-Acuña; Serra, 2016) and hatch when favorable conditions resume. However, earlier studies that sought to evaluate the chemical substances that affect hatching patterns usually tested substances which are highly toxic and under unrealistic high concentrations (Hanazato 1998, 2001). The toxic potential of DOC is low in comparison with most other compounds currently tested, as it is naturally found in different concentrations ranging from low levels up to 300 mg/l (Blodau et al. 2004; Sobek et al. 2007).
Interestingly, DOC effects on hatching patterns were non-linear, as intermediate concentrations (50 mg/L DOC) determined higher hatchling numbers and lower time to hatching. This finding agrees with earlier studies on DOC effects on active cladocerans. Overall, intermediate DOC concentrations are more beneficial than extreme values (Suhett et al. 2011). For active organisms, it has been suggested that intermediate DOC concentrations benefit organisms by inducing genes related to stress responses (Steinberg et al. 2010). The higher hatching in intermediate concentrations of DOC can possibly occur because light attenuation promotes an optimal light stimulus in eggs. Moreover, the toxic effect of DOC would only occur in high concentrations, with an intermediate concentration to be inert or little toxic. Thus, the light attenuation process can be a dominant factor in resurgence processes mediated by DOC mainly in intermediate concentrations.
DOC of any source with high HS concentration attenuates light intensity. Factors other than DOC in natural systems which can affect light intensity in bottom sediments where resting eggs accumulate are depth, inorganic turbidity and algal biomass (Scheffer, 1999; Henley et al., 2000; Donohue and Garcia Molinos, 2009). Resting eggs deposited in the sediment receive less light as those factors increase, but DOC is commonly acknowledged as the main factor of light attenuation in aquatic systems (Morris et al., 1995; Karlsson et al., 2009).
Changes in DOC concentrations can affect the reestablishment of zooplankton populations by changing the hatching success and time to hatching. Resting egg banks are the main contributors for community resilience after disturbances in natural environments (Hairston et al., 2000; Brendonck and De Meester, 2003). DOC effects can change the relative abundances of species which are more sensitive to light and create a numeric advantage in individuals able to reproduce fast in the water column.
This is the first study assessing how DOC affects zooplankton resting eggs and provided an effective separation of physical and chemical effects of DOC. Our study suggests that DOC mainly affects hatching patterns through light attenuation, and this effect can influence the number of hatchlings and the time to hatching. The increase of DOC in freshwater systems worldwide (water brownification) will affect hatching patterns and may have important ecological consequences in the future. Moreover, the effects of DOC in resting eggs of varying taxa and at the community level remains to be evaluated, as species may differ in their responses to varying DOC concentrations.