Scientists estimate biomass of invertebrates to evaluate wetland management practices, estimate energetic carrying capacity for wildlife, assess habitat condition and disturbance, and quantify ecosystem services. For waterfowl and other waterbirds in North America, carrying capacity in migratory and wintering regions is estimated using food density, of which invertebrates can be a significant component. However, we are not aware of previous literature that has described the effects of reagents used during core sample processing on invertebrate biomass and abundance. We tested the effects of hydrogen peroxide on aquatic invertebrates to determine whether a reagent used to disassociate soils during core sample processing biased estimates of biomass and abundance. Wet masses of chironomid larvae were less ( = 23.5% loss) in samples exposed to hydrogen peroxide than those exposed only to tap water and biomass decreased approximately 2.9% with each minute of exposure time. Dry mass of larvae was less in samples exposed to hydrogen peroxide than in those exposed only to tap water ( = 2.5% loss), but we did not detect an effect of exposure time on mass lost. Hydrogen peroxide did not influence the abundance of macro- or microinvertebrates in test samples. Thus, bias associated with dry mass estimates of invertebrates from core samples treated with hydrogen peroxide is likely minimal in terms of application in energetic carrying capacity models. However, use of hydrogen peroxide during core sample processing may cause significant bias if biomass estimates are based on wet mass.

Scientists estimate biomass of invertebrates to evaluate wetland management practices (Hagy and Kaminski 2012), estimate food availability for wildlife (Straub et al. 2012), indicate habitat conditions and disturbance in natural systems (Paoletti and Bressan 1996; Anteau and Afton 2008), and quantify ecosystem services (DeSzalay and Resh 2000; Manley et al. 2005; Prather et al. 2013). Aquatic invertebrates are used as food by waterbirds throughout their annual cycle and are particularly important during spring migration, feather molt, and the breeding season as a source of protein, lipids, and essential amino acids (Bellrose 1980; Loesch and Kaminski 1989; Richardson and Kaminski 1992; Anderson et al. 2000; McKinney et al. 2004). Often, scientists include aquatic invertebrate biomass with seeds, tubers, and other plant materials in estimates of food density that are used to determine energetic carrying capacity for waterbirds. Energetic carrying capacity can be used as a surrogate measure of wetland quality and to determine habitat conservation objectives for waterbirds (Soulliere et al. 2007; Gray et al. 2013; Williams et al. 2014).

Scientists often estimate biomass and density of aquatic invertebrates from benthic core samples, but this process can be expensive and time-consuming. Scientists often use solvents to dissolve substrates and increase efficiency of sample processing (Kross et al. 2008; Hagy et al. 2011; Straub et al. 2012). A common method used to increase efficiency of processing core samples from clay-laden soils is treatment with 3% hydrogen peroxide (H2O2; Kross et al. 2008; Havens et al. 2009; Hagy and Kaminski 2012; Olmstead et al. 2013). Hydrogen peroxide disassociates soil particles and facilitates separation of seeds, tubers, and invertebrates from the soil (Bohm 1979). However, no previous studies have evaluated the effects of H2O2 exposure on abundance or biomass of aquatic invertebrates. If exposure time has negative effects on aquatic invertebrates, then previous studies that used these techniques may have underestimated abundance and biomass, and this bias could subsequently affect estimates of energetic carrying capacity and result in overestimates of habitat requirements by conservation planners (Soulliere et al. 2007).

We tested the effects of H2O2 used to disassociate soils during core sample processing on aquatic invertebrate biomass and abundance. We exposed one common macroinvertebrate taxa (Chironomidae, larvae) and three microinvertebrate taxa (Cladocera, Ostracoda, Copepoda) to H2O2 and tap water (H2O; control) for different times to determine whether length of exposure influenced biomass or abundance. Our hypothesis was that aquatic invertebrate biomass and abundance would not be related to exposure time to H2O2 as has been assumed by previous researchers.

Chironomid larvae were collected from Pool 19 of the Mississippi River and Rice Lake of the Illinois River by using a 10-cm-deep core sampler during March 2013. Samples were preserved in 10% formalin for <60 days until trials began consistent with previous studies (Donald and Paterson 1974; Smith et al. 2012). We removed chironomid larvae (hereafter, larvae) from preserved core samples by hand and gently rinsed each in H2O to remove all residual soil and preservative. Experimental samples of 20 larvae were then placed on a paper towel, gently blotted dry, and weighed to the nearest 0.1 mg to obtain a pretreatment wet mass. Experimental samples were then placed in a petri dish containing either H2O2 (treatment) or H2O (control) for 2 min (n = 3), 5 min (n = 3), 10 min (n = 3), 180 min (n = 2), 540 min (n = 2), and 1,440 min (n = 2). Once samples had been soaked for the allotted time, they were placed into a #35 sieve (500 μm) and rinsed with H2O to simulate core sample processing procedures (Hagy et al. 2011). Samples were next placed on a paper towel, gently blotted dry, and weighed to the 0.1 mg to obtain a posttreatment wet mass. Finally, samples were placed in a drier at 60°C for 24 h and then weighed to the nearest 0.1 mg to obtain a posttreatment dry mass.

We obtained microinvertebrates from the taxa Cladocera, Ostracoda, and Copepoda from core samples obtained and processed similarly as described above. We exposed 10 individuals of each taxa to either H2O2 (treatment; n = 2/taxon) or H2O (control; n = 2/taxon) for 5 min. We used only the 5-min exposure time because results of the previous experiment demonstrated possible mass loss after this time, and alternative sorting techniques could be used to reduce exposure time to approximately 5 min. Once samples had been soaked for the allotted time, they were placed into a #35 sieve (500 μm), rinsed with H2O, poured into a white invertebrate sorting tray, and enumerated. We could not estimate biomass of small numbers (<300 individuals) of microinvertebrates by using available balances (≥0.1 mg) and isolating large numbers of microinvertebrates for this experiment was not practical. Because microinvertebrates are typically omitted from food biomass estimates for waterbirds or enumerated and multiplied by a constant to derive a mass estimate, we determined that estimating effects of H2O2 on abundance was the most practical and cost-effective procedure.

We tested for homogeneity of variances, examined residuals to ensure normal distribution, and designated α = 0.05 for all tests. We used separate paired t-tests to compare percentage mass loss between samples soaked in H2O2 and those soaked in H2O by using wet masses and dry masses. In separate analyses, we used analysis of variance (Proc GLM, SAS version 9.3) to test for effects of exposure time (independent variable) on differences in wet mass and dry mass (dependent variables) of samples soaked in H2O2. Similarly, we tested for effects of exposure time (independent variable) on differences in wet mass and dry mass (dependent variables) of samples soaked in H2O (control). Post hoc Tukey tests were used to compare means between time intervals if main effect of time was significant in any analysis.

Mass loss (wet) of larvae was greater (t14 = 5.7; P < 0.01) in samples exposed to H2O2 than in those exposed only to H2O ( = 23.5% loss, range = 0.8–50.3%; Table 1). Loss of wet mass in samples exposed to H2O2 also increased with exposure time (F5,9 = 22.8; P < 0.01; Figure 1). Biomass decreased linearly by 2.9% each minute of exposure time during the first 10 min, but subsequently the rate declined to 1% every 90 min. Mass loss (dry) of larvae was also greater (t14 = 2.3; P = 0.04) in samples exposed to H2O2 than in those exposed only to H2O ( = 2.6% loss, range = −4.8–8.6%), but we did not detect an effect of exposure time on mass lost after drying samples (F5,9 = 1.9; P = 0.19). Neither wet mass loss (F5,9 = 0.4; P = 0.84) nor dry mass loss (F5,9 = 2.9; P = 0.08) of larvae soaked in H2O was related to time of exposure. We noted that discoloration and significant degradation of larvae became visually apparent after 5 min of soaking in H2O2, but we did not observe any loss of individual macroinvertebrates in either treatment or control groups.

Table 1.

Wet and dry mass of Chironomidae spp. larvae exposed to water (H2O; control group) and hydrogen peroxide (H2O2) for differing times during spring 2013 at the Forbes Biological Station, Havana, Illinois.

Wet and dry mass of Chironomidae spp. larvae exposed to water (H2O; control group) and hydrogen peroxide (H2O2) for differing times during spring 2013 at the Forbes Biological Station, Havana, Illinois.
Wet and dry mass of Chironomidae spp. larvae exposed to water (H2O; control group) and hydrogen peroxide (H2O2) for differing times during spring 2013 at the Forbes Biological Station, Havana, Illinois.
Figure 1.

Percent mass lost (wet) after chironomid larvae (n = 20/sample) were soaked in 3% hydrogen peroxide (H2O2) and tap water (H2O) for different durations during spring 2013 under controlled conditions at the Forbes Biological Station, Havana, Illinois. Different letters indicate difference (α = 0.05) in exposure time by using a 1-way analysis of variance and a post hoc Tukey comparison of means.

Figure 1.

Percent mass lost (wet) after chironomid larvae (n = 20/sample) were soaked in 3% hydrogen peroxide (H2O2) and tap water (H2O) for different durations during spring 2013 under controlled conditions at the Forbes Biological Station, Havana, Illinois. Different letters indicate difference (α = 0.05) in exposure time by using a 1-way analysis of variance and a post hoc Tukey comparison of means.

Close modal

We recovered 100% of individuals from all microinvertebrate taxa exposed to H2O2 for 5 min and 1.5 d, and there was no visual evidence of degradation. We recovered 96.7% of individuals exposed to H2O. The two unrecovered microinvertebrates were both Cladocera from a single trial, and they apparently disintegrated during the trial period (Table 2).

Table 2.

Abundance of microinvertebrate taxa exposed to water (H2O; control group) and hydrogen peroxide (H2O2) for differing times during spring 2013 at the Forbes Biological Station, Havana, Illinois.

Abundance of microinvertebrate taxa exposed to water (H2O; control group) and hydrogen peroxide (H2O2) for differing times during spring 2013 at the Forbes Biological Station, Havana, Illinois.
Abundance of microinvertebrate taxa exposed to water (H2O; control group) and hydrogen peroxide (H2O2) for differing times during spring 2013 at the Forbes Biological Station, Havana, Illinois.

Previously, researchers have demonstrated the effects of sample gear and acquisition (Behney et al. 2014; Ringelman et al. 2015), preservation method (Salonen and Sarvala 1985), sorting (Hagy et al. 2011), subsampling (Stafford et al. 2011), and other factors (Williams et al. 2014) on estimating biomass of waterfowl foods from core samples, but data were previously unavailable on the effects of processing solvents on biomass estimates. Contrary to our original hypothesis, we found a negative effect on wet biomass of exposing larvae to H2O2, but consistent with our hypothesis we found no substantial effect of H2O2 on dry mass during typical periods of exposure to H2O2 (<5 min). Similarly, Donald and Paterson (1977) showed differences as great as 47% in wet masses of larvae across different preservatives and solvents. Thus, researchers using dry biomass from soft-bodied macroinvertebrates exposed to H2O2 may not incur large biases (<3%), and effects observed are likely significantly less than the effects of using preservatives (Donald and Patterson 1974; Salonen and Sarvala 1985) and other potential sources of bias (Williams et al. 2014).

Consistent with our original hypothesis, we failed to detect effects of exposure to H2O2 on abundances of microinvertebrates. Although larvae seemed to physically degrade after 5 min of exposure to H2O2, no such effects were visually evident in aquatic microinvertebrates examined. Although we did not test for the effects of exposure time on abundances of all microinvertebrate taxa, we noticed no deleterious effects of H2O2 on Copepoda abundance after 1.5 d of exposure and we assume the results would be similar for other taxa. Moreover, macroinvertebrates did not dissolve or disappear in H2O2, even after noticeable discoloration when exposed for 24 h. Because we selected relatively soft-bodied taxa and assume that more robust taxa would show even less degradation (e.g., Dytiscidae, Gyrinidae, Notonectidae), use of H2O2 to increase processing efficiency of benthic core samples is unlikely to bias abundances of micro- or macroinvertebrates from core samples.

If even slight biases in biomass need to be avoided by future researchers using core samples, we suggest modifying field sampling methodologies to reduce soils present in laboratory samples. We suggest using a sieve bucket with a ≥500-μm bottom screen (Wildco, Yulee, FL) to extensively rinse samples in the field before laboratory processing. Samples prerinsed in the field immediately after collection are easier to transport, require less preservative, and require less H2O2 and other solvents during laboratory processing. We also advocate a two-step sorting process whereby large and conspicuous invertebrates are removed before using H2O2. Removal of large and conspicuous invertebrates before using H2O2 means that individuals and taxa comprising most of the biomass will be removed before the use of H2O2 and biases will be minimized without dramatically affecting processing time (Osborn 2015). In addition, we noticed anecdotally that soils within core samples that had been frozen tended to disassociate more readily than fresh core samples, but we did not experimentally test this hypothesis; freezing core samples may result in degradation of invertebrates rapidly after thawing (Salonen and Sarvala 1985).

Previous studies that have estimated invertebrate biomass from core samples by using H2O2 as a reagent may have slightly underestimated biomass if exposure was <5 min and dry mass was used. However, these biases may be limited when invertebrate biomass makes up a small portion of the overall biomass estimates used in energetic carrying capacity models (e.g., seeds plus tubers plus invertebrates). For example, Hagy and Kaminski (2012) reported invertebrate biomass in moist-soil wetlands made up <1% of estimates of food for waterfowl during winter in the Mississippi Alluvial Valley. Similarly, Osborn (2015) reported biomass of invertebrates was <5% of waterfowl foods in benthic samples from moist-soil wetlands, but invertebrates made up approximately 88% of foods in mudflats, indicating habitat-specific differences in possible effects of bias from invertebrate biomass estimates. Thus, bias in energetic carrying capacity estimates resulting from core samples treated with H2O2 may vary according the proportion of soft-bodied invertebrates in samples, which varies by habitats of waterbirds (e.g., moist-soil wetlands, mudflats; Osborn 2015). Significant bias may exist if biomass estimates are based on wet masses and alternative methods (e.g., length–weight models) may be better suited to estimate biomass from soft-bodied invertebrates exposed for long periods to H2O2 (McKinney et al. 2004). Scientists should avoid using H2O2 for long-exposure times or adjust biomass estimates accordingly if biomass estimates are based on wet mass.

We thank staff and technicians from the Forbes Biological Station for assistance with this project. Funding and support was provided by the Illinois Department of Natural Resources through Federal Aid in Wildlife Restoration (W-43-R) and the Illinois Natural History Survey through the Prairie Research Institute and the University of Illinois at Urbana-Champaign. Any opinions, findings, conclusions, or recommendations expressed are those of the authors and do not necessarily reflect the views of the Illinois Department of Natural Resources or other collaborators.

Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

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Author notes

Citation: Hagy HM, McKnight JS. 2016. Effects of hydrogen peroxide as a core sample processing solvent on invertebrate biomass. Journal of Fish and Wildlife Management 7(2):444–448; e1944-687X. doi: 10.3996/042016-JFWM-032

The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service.