333 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 A B S T R A C T Knowing about the ecological aspects involved in the commercial breeding of aquatic organisms becomes an important tool to make aquaculture more productive and less impactful. Thus, periphyton taxon composition and biomass on different substrates and the influence of these on water quality and growth parameters of Nile tilapia fingerlings were examined. An experiment with three treatments (substrates for growth of periphyton: geomembrane, polyethylene terephthalate (PET), and bamboo) and a control (without substrate), each with five replicates, was conducted in a greenhouse with controlled aeration and temperature. Each mesocosm was populated with ten tilapia fingerlings with an average weight of 2.3 g for 30 days. Water quality parameters were not significantly different among treatments but remained within that established by the environmental legislation. In all treatments, 36 periphyton taxa were observed. The bamboo substrate was the most diverse, which could be attributed to the fact it was a natural substrate. Regarding fish growth, there was a significant difference among the treatments, with the PET treatment having a higher condition factor (kn). The bamboo substrate was good for colonization concerning alga diversity; however, fish in the PET treatment and control exhibited higher performance and algae consumption values, respectively. Keywords: periphytic algae; ecologic aquaculture; commercial fish feeding. R E S U M O Conhecer os aspectos ecológicos que estão envolvidos na criação comercial de organismos aquáticos é ferramenta importante para tornar a aquicultura mais produtiva e menos impactante. Assim, este trabalho avaliou a composição de táxons, a biomassa do perifíton em diferentes substratos de fixação, a influência destes na qualidade de água e o desempenho zootécnico de alevinos de tilápia-do-nilo. Um experimento com três tratamentos (diferentes substratos para crescimento do perifíton: geomembrana, polietileno tereftalato [PET] e bambu) e um controle (sem substrato), com cinco repetições cada, foi montado em uma casa de vegetação com temperatura e aeração controladas. Cada mesocosmo foi povoado com dez alevinos com peso médio de 2,3 g, por 30 dias. Os peixes foram alimentados com ração comercial, cuja taxa de arraçoamento foi 30% menor do que a recomendada pelo fabricante, para estimular o consumo de perifíton. Os parâmetros de qualidade da água não foram significativamente diferentes entre os tratamentos, mas mantiveram- se em conformidade com o estabelecido pela legislação brasileira. Em todos os tratamentos, foram encontrados 36 táxons de perifíton. O substrato bambu foi o mais diverso, considerando-se o número de táxons encontrados, o que pode ser atribuído ao fato de ser ele de origem natural. O substrato geomembrana apresentou crescimento perifítico superior aos demais, com maior quantidade de biomassa final. O único resultado significativamente diferente entre os tratamentos foi o tratamento PET com maior valor de fator de condição (kn), que indicou melhores condições de bem-estar dos peixes. O substrato bambu mostrou-se bom para colonização em relação à riqueza de algas perifíticas, todavia os peixes dos tratamentos PET e controle mostraram melhores valores de desempenho e consumo de algas, respectivamente, em relação aos outros tratamentos avaliados. Palavras-chave: algas perifíticas; aquicultura ecológica; alimentação de peixes comerciais. Contributions of the periphyton to the growth of Nile tilapia (Oreochromis niloticus) fingerlings on different fixation substrates: an ecological approach Contribuições do perifíton no crescimento de alevinos de tilápia-do-nilo (Oreochromis niloticus) em diferentes substratos para fixação: uma abordagem ecológica Ynaê Paula Schroder Rosa1 , Márcia Regina Russo1 , Luis Antônio Kioshi Aoki Inoue2 , Lidiany Doreto Cavalcanti3 1Universidade Federal da Grande Dourados – Dourados (MS), Brazil. 2Empresa Brasileira de Pesquisa Agropecuária – Dourados (MS), Brazil. 3Universidade Estadual de Maringá – Maringá (PR), Brazil. Correspondence address: Ynaê Paula Schroder Rosa – Rua Apolo, 68 – Jardim Jacy – CEP: 79006-760 – Campo Grande (MS), Brazil. E-mail: ypschroder@gmail.com Conflicts of interest: the authors declare no conflicts of interest. Funding: Conselho Nacional de Desenvolvimento Científico e Tecnológico. Received on: 10/22/2021. Accepted on: 04/03/2022. https://doi.org/10.5327/Z2176-94781253 Revista Brasileira de Ciências Ambientais Brazilian Journal of Environmental Sciences Revista Brasileira de Ciências Ambientais Brazilian Journal of Environmental Sciences ISSN 2176-9478 Volume 56, Number 1, March 2021 This is an open access article distributed under the terms of the Creative Commons license. https://www.google.com.br/search?q=Oreochromis+Niloticus&spell=1&sa=X&ved=0ahUKEwjI7__DgrzNAhXKPj4KHV7XD5QQBQgbKAA https://orcid.org/0000-0002-1536-0442 https://orcid.org/0000-0003-1526-7130 https://orcid.org/0000-0002-9582-7654 https://orcid.org/0000-0001-8846-9417 mailto:ypschroder@gmail.com https://doi.org/10.5327/Z2176-94781253 http://www.rbciamb.com.br http://abes-dn.org.br/ https://creativecommons.org/licenses/by/4.0/ Rosa, Y. P. S. et al 334 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 Introduction Sustainable aquaculture requires innovation and good environ- mental management, whereas aquaculture productivity depends on the project and an appropriate method to reduce its impact on the aquatic environment (Putro et al., 2020). Thus, knowing the ecological aspects that are involved in the commercial breeding of aquatic organ- isms becomes an important tool to make aquaculture more productive and less impactful. In fish farming, as well as in other aquatic ecosystems, different aquatic organisms grow together along with the fish. These elements, constantly invisible to the fish farmer, compete for space, food, oxygen, and nutrients, cause disease, and also contribute to the ecological bal- ance of the production environment (Russo et al., 2021). Periphyton is a universally recognized term that designates all organisms attached to a submerged substrate, usually dominated by single-cell, colonial, or filamentous photosynthetic organisms, both prokaryote and eukaryote (Inyang et al., 2018). According to Wetzel (1990), algae constitute approximately 90% of the periphyton, de- pendent on variation according to environmental conditions (other microorganisms comprise the remaining 10%). This characteristic has attracted the attention of researchers for its potential use in aquaculture. Most early-stage fish species, particularly herbivorous and om- nivorous fish, use these organisms as a protein, vitamin, and min- eral-rich food source (Pérez, 1992; Van Dam et  al., 2002). These attributes confer these organisms economic and environmental im- portance. In this respect, the periphytic community also plays an import- ant role in water quality and in the trophic state bioindicator thanks to its ability to accumulate substances (such as nutrients and pol- lutants) in its biomass (Neal et al., 1967; Tedeschi and Chow-Fraser, 2021). This feature allows the periphyton to operate as a biofilter, directly and positively influencing nutrient cycling (Azim et  al., 2001, 2004). Feed and fertilizers are used during the production cycle in the normal daily operation of fish farms, but they may be expensive, and their dosage is often poorly calculated. An excess of these prod- ucts in the water leads to an incomplete utilization by the fish, and as waste, they contribute to eutrophication and compromise water quality and fish cleanliness (Carballo et  al., 2008). Therefore, the use of substrates that increase the area of periphyton growth in fish farming tanks can be an effective tool to reduce the nutrient load stemming from production residues (Biswas et  al., 2018), is a nat- urally available food source, and, consequently, reduces the use of artificial diet, thereby alleviating production costs (particularly for small-scale producers) (Garcia et al., 2017). In this regard, experiments that evaluate the contribution of the periphyton to the initial growth phases of commercial fish, the pos- sible lower impacts on water quality, and optimization of integrated systems are fundamental to improving the management tools that minimize environmental and economic impacts caused by the ex- clusive use of artificial diets, particularly in extensive production. The goal of this study was to evaluate the taxon and biomass composition of the periphytic community on different fixation substrates, as well as their influence on water quality and on the growth parameters of Nile tilapia (Oreochromis niloticus) with the hypothesis that the periphyton substrates in the mesocosms would contribute to the zootechnical performance of the fish. Material and Methods The experiment was carried out in a greenhouse facility locat- ed at EMBRAPA Agropecuária Oeste, in Dourados, state of Mato Grosso do Sul, for 30 days between April and May 2017. Light (50% shade), temperature (air conditioning), and constant aeration of the water were controlled to provide an environment for the experi- ment that was almost the same as the natural one. A total of twenty 100 L polyethylene boxes were used, submitted to constant aeration, and distributed in a completely randomized experi- mental design with four treatments and five replicates: control (with- out periphyton-fixating substrate), bamboo substrate, geomembrane substrate High-Density Polyethylene (HDP — Thickness: 5  mm), and polyethylene terephthalate (PET) bottle substrate. The substrates were chosen because of the ease of access and handling, low cost, and the presence of mechanical action-resistant surfaces for scraping. In each treatment, a 30 × 40 (cm) rectangular structure with a base of galva- nized wire covered with antirust paint was employed for the fixation of the different periphyton growth substrates. This set occupied approx- imately 10% of each box’s total area. The substrates contained within the wireframe base measured 30 × 5 cm and were kept fully immersed. The set was attached to one of the edges of each tank. The fish were purchased from a local fish farm and acclimatized for 7 days. Each box was stocked with ten fish, with an average weight of 2.3 ± 0.01  g, fed with commercial feed (a 40% crude protein feed at the beginning of the experiment, followed by a 4 mm pelleted feed with 36% crude protein, according to the growth of the fish) at a dos- age corresponding to 70% of the manufacturer’s recommendations to stimulate periphyton consumption. The total daily feed amount was 10% of the fish biomass. The feeding frequency was four times per day at 7 a.m., 11 a.m., 1 p.m., and 4 p.m. The bottom of each box was siphoned to remove excess waste. Approximately 40% of the water volume was replenished every 2 days. Dissolved oxygen and temperature values were measured daily using a YSI model 55 probe. Once a week, pH, electrical conductivity, and dissolved solids were measured using a Horiba U50  multiparameter probe. Nitrite, ammonia, and orthophosphate concentrations, plus hardness and alkalinity, were measured using Alfakit colorimetric kits. All of these parameters were then compared with CONAMA 357/05, the current legislation available. Contributions of the periphyton to the growth of Nile tilapia (Oreochromis niloticus) fingerlings on different fixation substrates: an ecological approach 335 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 The structures containing the substrates were removed weekly to scrape off an area of approximately 10% of the total area with a glass slide. The species composition was qualitatively analyzed by Transeau fixation. The taxa of the different substrates were identified under an optical microscope using a 3  mL aliquot per replicate by preparing temporary slides for binocular optical microscopy, Olympus CX41 (Bicudo and Menezes, 2006). After 30 days, a biometric analysis was performed (weight, and to- tal and standard length). For this procedure, ten fish of each box were euthanized using the benzocaine lethal method (immersion in pH 7-buffered solution), according to the guidelines of Normative Reso- lution 37/2018 — National Council for Animal Experimentation Con- trol (CONCEA), gutted, and their stomachs were separated for content analysis. Each stomach was immersed in 4% formaldehyde solution in a labeled glass vial for further dietary analysis. This research was previously certified by the committee on animal research and ethics of Universidade Federal da Grande Dourados un- der protocol number 51/2016 and approved in a meeting held on No- vember 18, 2016. In addition to final weight averages, the following data were calcu- lated per treatment. According to Inoue et al. (2014), the weight gain values were obtained by the formula: Final average weight (g) – Initial average weight (g), and the daily weight gain by Daily Weight Gain: Weight gain (g)/ number of days. The Relative (Kn) or Le Cren condition factor of fish in each treatment was calculated using Kn = W observed/W expected, where W observed was the weight of each individual and W expected, the weight determined by the weight-length curve (Le Cren, 1951; Araú- jo et al., 2011). To evaluate the existence and proportion of periphyton consumption in each treatment, the stomach contents of the fish were analyzed by the frequency of occurrence method (%FO) using an optical microscope and a Sedgewick – Rafter counting chamber; the number of individuals that con- sumed a given item (feed, different periphyton taxa) was expressed as the percentage of total examined fish with stomach contents (Bowen, 1992). Thus, the number of times each item occurred was treated as a percentage of the total number of occurrences of all items (Hahn and Delariva, 2003). The effect of the substrates on water quality was tested via a one- way analysis of variance (ANOVA), followed by a Tukey’s test (p < 0.05) evaluating the physical-chemical parameters. The weight and length data were used to calculate the condition factor followed by a one-way ANOVA to check for significant differences between the factors per treatment. The same biometric data and statistical tests were used to compare all performance results using SigmaStat 4.0 software. With data on the periphyton taxa composition by substrate, a Cluster analysis was performed to evaluate taxa similarity vs. collection days. For stomach contents data, Permutation Multivariate Analysis of Variance (PERMANOVA) with the Bray-Curtis Coefficient (999 permutations) (Anderson, 2005) was applied to verify the significant differences in the composition of the diet in the different treatments. In addition, the Indi- vidual Indicator Value (IndVal) was calculated to show the main fish diet items for each treatment. Principal Coordinate Analysis (PCoA) with Bray-Curtis distance was performed to visualize similarities or differenc- es in the diet composition of fish between treatments. For the aforemen- tioned analyses, software R (R Core Team, 2003) was employed. Results and Discussion Water quality complied with the standard established by the CONA- MA 357/05 resolution for class 2 water bodies and is shown in Table 1. The parameters remained constant throughout all treatments. The average values were calculated considering the entire experimental period and none of the calculated average values showed a significant difference. Table 1 – Means and standard deviations of the physical-chemical parameters of water during the 30 days of the experiment in treatments with different periphyton growth substrates. Control Geomembrane Bamboo PET NS Dissolved oxygen (mg.L-1) 6.96 ± 0.7 6.97 ± 0.58 6.75 ± 0.94 7.0 ± 0.49 0.84 Temperature (°C) 22.08 ± 0.87 22.1 ± 0.94 22.1 ± 0.84 22.0 ± 0.82 0.99 pH 7.14 ± 0.67 7.05 ± 0.61 7.06 ± 0.6 7.16 ± 0.67 0.99 Alkalinity (CaCO3 mg.L -1 ) 61.28 ± 24.6 55.16 ± 27.5 67.44 ± 11.6 59.0 ± 19.4 0.97 Hardness (CaCO3 mg.L -1) 67.36 ± 10.5 70.68 ± 10.6 60.2 ± 20.0 68.0 ± 12.4 0.93 Total ammonia (mg.L-1) 0.97 ± 1.11 0.89 ± 1.24 1.07 ± 1.34 0.83 ± 1.13 0.98 Orthophosphate (PO4 mg.L -1) 0.16 ± 0.188 1.24 ± 0.54 1.01 ± 0.61 0.94 ± 0.57 0.64 Nitrite (NO3 mg.L -1) 0.16 ± 0.18 0.16 ± 0.19 0.15 ± 0.18 0.16 ± 0.19 > 1.0 Conductivity (μS.cm-²) 0.422 ± 0.02 0.421 ± 0.02 0.424 ± 0.03 0.422 ± 0.02 0.99 Total dissolved solids (mg.L-1) 0.274 ± 0.01 0.273 ± 0.01 0.275 ± 0.02 0.274 ± 0.01 0.96 Redox Potential (ORP) 191.16 ± 48.6 159.8 ± 93.9 188.5 ± 50.5 193.4 ± 45.9 0.99 NS: significance level. http://S.cm Rosa, Y. P. S. et al 336 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 In this study, substrates occupied approximately 10% of the total me- socosm, which may have been insufficient for the growth of periph- yton biomass to have a beneficial effect on water quality parameters. Previous literature (Keshavanath et al., 2001) reported low values of dissolved oxygen in treatments with natural periphyton growth substrates when compared to an artificial substrate, in that case, the PVC pipe substrate. Azim et  al. (2004) showed that the presence of periphyton decreased the nitrogen and total ammonia concentra- tions, operating as a biofilter in the tanks. In contrast, Andrion (2014) did not observe significant differences between treatments, probably because the periphyton failed to develop because of the presence of suspended particulate material, where the main water parameters re- mained constant. Thirty-five taxa were identified in the periphytic community, the majority of which were in the algae group (23) — mainly Chlorophy- cea, Oedogoneophycea, and Diatomaceae. Other taxa included rotifers (2), fungi (1), copepods (1), protozoa (6), and insects (1). Rotifer eggs were also found, which were classified as a type of food item taxon. The richness of taxa vs. the type of substrate was demonstrated through a similarity analysis (Figure 1). The dendrogram illustrates which substrates were most similar concerning taxa composition per collection (Figure 1). In the artifi- cial substrates (PET and geomembrane), there was a higher similarity among taxa, probably because of the similar substrate nature (smooth surfaces). Tortolero et  al. (2015) described a similar result, wherein natural substrates exhibited a more diverse composition and higher biomass compared to artificial substrates. The number of taxa found increased in all treatments with time (Figure 2). The increasing colonization was visible, particularly in the bamboo and geomembrane substrates. This result may be a consequence of the periphytic succession pro- cess, as described in studies that evaluated the growth and dynamics of periphyton succession in natural and/or artificial substrates (Siqueira and Rodrigues, 2009; Felisberto and Rodrigues, 2012). Several factors can influence this behavior in natural environments, including the rainfall regime and the roughness of the substrate available for periph- ytic community growth (Vercellino and Bicudo, 2006). Bergey (2005) demonstrated that the biomass of periphytic algae increased with the number of cracks in the substrate. Studies such as one by Osório et al. (2019) showed that the colo- nization of the periphytic community differed according to the abili- ty of each species to colonize substrates of small or large complexity. Murdock and Dodds (2007) obtained higher values of chlorophyll and higher diversity of algae in more rugged substrates, suggesting that the  larger surface area available with rough substrates contributes to the growth and diversity of species in the community, as does the com- plexity of the substrate. There were significant differences in diet composition (consumed items), between treatments (PERMANOVA F = 4.5382, p = 0.01), and the IndVal (Table 2) showed that the indicator items of the bamboo treatment were the algae Monoraphidium sp, Pennales, and Selenas- trum sp; for geomembrane, there were no indicator items and the fish ingested higher amounts of feed when compared to the other systems evaluated herein. In the PET treatment, the highlighted items were ro- tifer, and resistance egg groups; in the control group, Scenedesmus sp. was the indicator item. Ordering analysis (PCoA) explained the 42% data variation, and important segregation was observed among treatments containing substrates and the control treatment (Figure 3). In both, the analysis of tilapia stomach contents showed a diversified diet, rich in natural foods. Periphytic algae taxa were consumed, as well as planktonic al- gae of the Scenedesmus genus (Appendix 1), which are one of the most common phytoplanktonic components present in freshwater bodies (Bicudo and Menezes, 2006). These algae were the most consumed nat- ural item by fish in the control treatment, possibly because of the ab- sence of substrate that allows the growth of the periphytic community. Figure 1 – Cluster dendrogram showcasing the similarity of taxa composition in each periphyton substrate treatment per week (1, 2, 3, 4, 5) throughout the 30-day experiment. Geo: geomembrane. Figure 2 – Relationship between the number of periphyton taxa vs. substrate in the three treatments (bamboo, PET, and geomembrane). Contributions of the periphyton to the growth of Nile tilapia (Oreochromis niloticus) fingerlings on different fixation substrates: an ecological approach 337 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 Loures et  al. (2001) observed that for tilapia from fish farms fed mainly on feed, at certain times, the consumption of phytoplank- ton was almost equal to that of the feed provided by the researcher. The phytoplankton and periphytic communities require practically the same nutrients to grow, a higher consumption of phytoplanktonic al- gae (mainly from the Scenedesmus genus) in the control treatment sup- ports the hypothesis that the absence of a substrate in this treatment favored the development of phytoplanktonic algae. The choice of the item to be eaten by the fish is influenced by the availability of the resource, food preference, and prey size (Sipaú- ba-Tavares, 1993). Abimorad et  al. (2013) found that, while forag- ing, fish can select the food that better suits their nutritional needs, leaving unsuitable organisms in the substrate. Huchette et  al. (2000) observed a similar relationship between the species found in the arti- ficial substrates inserted in the culture environment with the species found in fish stomachs, corroborating what was observed in this study. Rivera Vasconcelos et al. (2018) observed that the magnitude of tilapia predation effect on the phytoplankton and zooplankton communities in tropical lakes is dependent on the biomass and structural size of both communities. Table 2 – Frequency of occurrence of food items found in the stomach contents of fish per treatment e IndVal significance values (p < 0.05). Items Bamboo Geomembrane PET Control Algae Monoraphidium sp. 4.54* 1.8 1.5 0.25 Scenedesmus sp. 1.95 1.6 0.65 16.5*** Chlorococcales 0 0.35 0.85 0.25 Desmodesmus sp. 0.55 0.2 0.04 0 Pennales 0.95** 0.3 0.1 0.05 Coelastrum sp. 1.6 1 4.45 2.1 Johannesbaptista sp. 0.4 0 0 0 Chroococcales 0.15 0.04 0 0 Selenastrum sp. 2*** 0.1 0.2 1 Stigeoclonium sp. 0.4 0 0.1 0 Bulbochaete 0.01 0.03 0 0 Binuclearia sp. 0.2 0.15 0.02 0 Gloeotila sp. 0.05 0.05 0 0 Oedogonium sp. 0.4 0 0 0 Oocystis sp. 0 0 0 0.1 Pseudoanabaena 0 0.88 0.15 0.05 Eudorina 0 0 0.04 0 Protozoa Suctoria 1.05 0.3 1.7** 0.6 Peritrichida 0.3 0.2 1.45 0 Synhymeniida 0 0.5 0 0 Rotifers Bdelloidea 3 2 6.2* 1.6 Eutoratoria 0.3 0.15 1.15 0.15 Other items Arcella 0.05 0.05 0 0 Scale 0.05 0 0.05 0 Rotifer eggs 1 0.3 1.4** 0.3 Chytridiomycetes 0.05 0.5 0.05 0 Feed 81 89.5 79.9 77.05 Total 100 100 100 100 *0.05; **0.01; ***0.001. Rosa, Y. P. S. et al 338 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 In the early stages, plankton is the main food source for most fish, in natural environments (Uys and Hecht, 1985) and, in fish farming, it has great importance as a supplementary item without additional costs, which can improve weight gain, condition factor, and immune responses to fluctuating weather conditions and pathogens (Uddin et al., 2009; Cavalcanti et al., 2021). In this study, there were no significant differences in zootechni- cal performance (p > 0.05) between the different treatments (Table 3). As compared to the Salazar-Torres et al. (2016) experiment, the authors argued that low weight gain stems from a failure to meet the tilapia’s energetic demands. Keshavanath et al. (2004), in a 75-day experiment, showed significant differences in the final weight of fish that fed on a system with bamboo substrates for periphyton growth, with fingerlings being able to predate the periphyton. The same may have occurred in our work, that is, the surface area for growth of the periphyton, as well as the duration of the experiment, may have been insufficient to pro- duce a significant effect on the weight and length of the fish. Still concerning zootechnical performance, PET treatment showed a higher condition factor, with significant differences (ANO- VA F = 275.5, p < 0.05) between treatments (Figure 4). The condi- tion factor is a way to quantify and compare the well-being, fat, or condition of the fish (Vazzoler, 1996). Variations in this indicator can be a result of several factors, including food availability, envi- ronmental quality, and the effects of pathogens (Bolger and Connol- ly, 1989; Ramos et al., 2013; Sarkar et al., 2013; Gouveia et al., 2020). Figure 3 – Principal coordinate analysis (PCoA) using Bray-Curtis dissimilarity of major food items consumed in the different treatments with substrates for periphyton growth and control. The items significantly consumed are in Table 2. However, it is essential to consider that PET is an artificial material in a natural environment. In a review, Miloloža et  al. (2021) observed that microplastic in freshwater sources may cause various adverse effects such as neurotox- icity, reproductive toxicity, oxidative stress, immunotoxicity, and a de- crease in photosynthetic efficiency in living organisms including algae, an important aspect of this study. Exploring the literature, Heindler et al. (2017) analyzed the effects of PET microparticles in copepods and one of the conclusions was that the prolonged exposure to microplastics had se- vere deleterious impacts on the population viability of these crustaceans. On the other hand, comparing the several types of plastic, their chem- ical characterization, and the ecotoxicological effects of chemical additive leaching, Capolupo et al. (2020) demonstrated that the PET plastic con- tained the lowest number and concentration of measured additives. Fish subjected to the PET treatment ingested a lower percent- age of feed compared to the other two treatments with substrates (79.9%). Moreover, animal items, such as rotifers and protozoa, were also consumed in higher proportions, a fact that may have influenced the better condition of fish (Gomiero et al., 2010; Araújo et al., 2011; Biswas et al., 2017). Table 3 – Zootechnical performance of tilapia fingerlings in all three substrate and control treatments, and significance represented by the p-value from the one-way ANOVA. Group Control PET Bamboo Geomembrane p-value Average final weight (g) 13.036 ± 3.54 13.786 ± 4.13 13.31 ± 3.32 13.52 ± 4.28 0.748 Weight Gain (g) 10.86 ± 4.01 11.96 ± 3.97 10.96 ± 4.18 11.37 ± 4.83 0.646 Daily weight gain (g/day/fish) 15.80 ± 5.84 17.11 ± 5.68 15.81 ± 6.03 16.40 ± 6.97 0.748 Specific Growth Rate (%/day) 1.17 ± 0.18 1.21 ± 0.17 1.16 ± 0.22 1.16 ± 0.27 0.684 Figure 4 – Fish condition factors per treatment. Contributions of the periphyton to the growth of Nile tilapia (Oreochromis niloticus) fingerlings on different fixation substrates: an ecological approach 339 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 Enhancement of natural food through the use of substrates in the ponds is a cheap alternative (Milstein et al., 2008; Barlaya et al., 2021). Additionally, in order to meet the growing demand and prevent the de- pletion of natural resources, aquaculture should become more sustain- able (FAO, 2020). Thus, substitutes for fish feed can reduce the impacts of aquaculture feed production (Boyd, 2015). In this sense, considering the consumption of periphyton by fish and the improvement in the observed condition factor of the PET substrate in this work, manip- ulating natural food can increase the productivity and efficiency of aquaculture production systems, making them more economically and environmentally sustainable. Conclusion The study evidenced a higher-taxon richness in the bamboo substrate, where a significant consumption of several groups of algae was also obser ved. There were no significant differences in water quality between treatments and control, neither there were positive effects of substrate use on these parameters, and this may be related to constant water exchange in the system. According to our results, the PET substrate treatment was a promising activity in this tilapia fingerling system. The type of periphytic organism that grows on the different substrates may be more important than diversity, providing a food supplement that improves the condition of the fish. Moreover, because this type of substrate is cost-free (as it is obtained from recycling), we emphasize the economic and environmental importance of the results obtained, considering the more sustainable produc- tion systems. We indicate the use of PET substrate in systems that are homologous to the one exposed in this study and recom- mend a larger occupation area for the substrate (around 20%) to increase the plankton available and possibly influence the final weight gain, concomitantly with ecotoxicological investigations to provide substantial information about the use of the PET ma- terial in these particular activities. This substrate has an expo- sure time for fish and cultured water that generally lasts around 6 to 8 months. Consequently, we believe that the short period of time this material can be exposed does not influence the health of the fish and water quality. Acknowledgements The authors extend their thanks to EMBRAPA, for the opportunity to conduct this experiment in their facilities. Contribution of authors: ROSA, Y. P. S.: Conceptualization; Data curation; Investigation; Methodology; Formal Analysis; Writing — original draft; Writing – Revision and Editing; RUSSO, M. R.: Conceptualization; Investigation; Writing — original draft; Writing – Revision and Editing; Supervision; Project Administration; Methodology; Formal Analysis; INOUE, L. A. K. A.: Conceptualization; Investigation; Writing — original draft; Writing – Revision and Editing; Supervision; Project Administration; Methodology; Formal Analysis; CAVALCANTI, L. D.: Conceptualization; Data curation; Investigation; Methodology; Formal Analysis; Writing — original draft; Writing – Revision and Editing. References Abimorad, E.G.; Garcia, F.; Romera, D.M.; Sousa, N.S.; Paiva-Ramos, I.; Onaka, E.M.; Campos, W.J.; David, L.H.C.; Tucci, A., 2013. Valor nutricional de perifíton em substrato de bambu na criação de tilápia em tanque-rede. Boletim do Instituto de Pesca, v. 37, (1), 31-38. Anderson, M., 2005. Permutational multivariate analysis of variance: a computer program. Department of Statistics, University of Auckland, Auckland, New Zealand. Andrion, B.C., 2014. Substratos artificiais melhoram a qualidade da água em sistema de cultivo multitróficos e multiespaciais? Master Dissertation, Centro de Aquicultura de Jaboticabal, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São Paulo. Araújo, C.C.; Flynn, M.N.; Pereira, W.R.L., 2011. Fator de condição e relação peso comprimento de Mugil curema valenciennes, 1836 (Pisces, mugilidae) como indicadores de estresse Ambiental. RevInter, v. 4, 51-64. Azim, M.E.; Wahab, M.A.; Biswas, P.K.; Asaeda, T.; Fujino, T.; Verdegem, M.C.J., 2004. The effect of periphyton substrate density on production in freshwater polyculture ponds. Aquaculture, v. 232, (1-4), 441-453. https://doi. org/10.1016/j.aquaculture.2003.08.010. Azim, M.E.; Wahab, M.A.; Van Dam, A.A.; Beveridge, M.C.M.; Huisman, E.A.; Verdegem, M.C.J., 2001. Optimization of stocking ratios of two Indian major carps, rohu (Labeo rohita Ham.) and catla (Catla catla Ham.) in a periphyton- based aquaculture system. Aquaculture, v. 203, (1-2), 33-49. https://doi. org/10.1016/S0044-8486(01)00602-0. Barlaya, G.; Umalatha, H.; Hegde, G.; Ananda Kumar, B.S.; Raghavendra, C.H., 2021. Growth performance, carcass composition, and digestive enzyme activity of Labeo fimbriatus in tanks provided with feed and periphyton substrate in two orientations. Journal of Applied Aquaculture, 1-12. https://doi.org/10.1080 /10454438.2021.1957054. Bergey, E., 2005. How protective are refuges? Quantifying algal protection in rock crevices. Freshwater Biology, v. 50, (7), 1163-1177. https://doi. org/10.1111/j.1365-2427.2005.01393.x. Bicudo, C.E.M.; Menezes, M., 2006. Gêneros de algas continentais do Brasil: chave para identificação e descrições. 2ª ed. Rima, 502 p. Biswas, B.; Das, S.K.; Mondal, I.; Mandal, A., 2018. Composite fish farming in West Bengal, India: redesigning management practices during the course of last five decades, International Journal of Aquaculture, v. 8, (12), 90-97. http:// doi.org/10.5376/ija.2018.08.0012. https://doi.org/10.1016/j.aquaculture.2003.08.010 https://doi.org/10.1016/j.aquaculture.2003.08.010 https://doi.org/10.1016/S0044-8486(01)00602-0 https://doi.org/10.1016/S0044-8486(01)00602-0 https://doi.org/10.1080/10454438.2021.1957054 https://doi.org/10.1080/10454438.2021.1957054 https://doi.org/10.1111/j.1365-2427.2005.01393.x https://doi.org/10.1111/j.1365-2427.2005.01393.x http://doi.org/10.5376/ija.2018.08.0012 http://doi.org/10.5376/ija.2018.08.0012 Rosa, Y. P. S. et al 340 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 Biswas, G.; Sundaray, J.K.; Bhattacharyya, S.B.; Shyne Anand, P.S.; Ghoshal, T.K.; Kailasam, M., 2017. Influence of feeding, periphyton and compost application on the performances of striped grey mullet (Mugil cephalus L.) fingerlings in fertilized brackishwater ponds. Aquaculture, v. 481, 64-71. http:// doi.org/10.1016/j.aquaculture.2017.08.026. Bolger, T.; Connolly, P.L., 1989. The selection of suitable indices for the measurement and analysis of fish condition. Journal Fish of Biology, 34, (2), 171-182. https://doi.org/10.1111/j.1095-8649.1989.tb03300.x. Bowen, S.H., 1992. Quantitative description of the diet. In: Nielsen, L.A.; Johnson D.L. (Eds.), Fisheries techniques. American Fisheries Society, Bethesda, pp. 325-336. Boyd, C.E., 2015. Overview of aquaculture feeds. Feed and Feeding Practices in Aquaculture, 3-25. https://doi.org/10.1016/b978-0-08-100506-4.00001-5. Brasil. Conselho Nacional do Meio Ambiente (Conama), 2005. Resolução Conama nº 357, de 17 de março de 2005. Diário Oficial da União. Capolupo, M.; Sørensen, L.; Jayasena, K.D.R.; Booth, A.M.; Fabbri, E., 2020. Chemical composition and ecotoxicity of plastic and car tire rubber leachates to aquatic organisms. Water Research, v. 169, 115270. https://doi.org/10.1016/j. watres.2019.115270. Carballo, E.; Van Eer, A.; Van Schie, T.; Hilbrands, A., 2008. Agrodok Agromisa. CTA, 96 pp. Cavalcanti, L.D.; Gouveia, E.J.; Carrijo-Mauad, J.R.; Russo, M.R., 2021. Effect of poultry litter as an organic fertilizer on water quality, parasitic abundance, and growth of Nile tilapia. Boletim do Instituto de Pesca, v. 47, e622. https:// doi.org/10.20950/1678-2305/bip.2021.47.e622. Conselho Nacional de Controle de Experimentação Animal, 2018. Diretriz Brasileira para o cuidado e a utilização de animais para fins científicos e didáticos. Conselho Nacional de Controle de Experimentação Animal, Brasília. Felisberto, S.A.; Rodrigues, L., 2012. Successional dynamic of the periphytic algal community in ecossistem lotic subtropical. Rodriguésia (online), v. 63, (2), 463-473. https://doi.org/10.1590/S2175-78602012000200018. Food and Agriculture Organization of the United Nations (FAO). The State of World Fisheries and Aquaculture, 2020. Sustainability in action. Food and Agriculture Organization of the United Nations, Rome. Garcia, F.; Sabbag, O.J.; Kimpara, J.M.; Romera, D.M.; Sousa, N.S.; Onaka, E.M.; Ramos, I.P., 2017. Periphyton-based cage culture of Nile tilapia: An interesting model for small-scale farming. Aquaculture, v. 479, 838-844. https://doi.org/10.1016/j.aquaculture.2017.07.024. Gomiero, L.M.; Villares Junior, G.A.; Braga, F.M.S., 2010. Length-weight relationship and condition factor for Oligosarcus hepsetus (Cuvier, 1829) in Serra do Mar State Park - Santa Virgínia Unit, Atlantic Forest, São Paulo, Brazil, Biota Neotropica, v. 10, (1), 101-105. https://doi.org/10.1590/S1676- 06032010000100009. Gouveia, E.J.; Cavalcanti, L.D.; Russo, M.R., 2020. Echinorhynchus gomesi Machado Filho, 1948 infecting the Patinga hybrid (♀ Piaractus mesopotamicus x ♂ Piaractus brachypomus) in fish farms in Mato Grosso do Sul, Brazil. Aquaculture Research, v. 51, (12), 5118-5124. https://doi.org/10.1111/ are.14850. Hahn, N.S.; Delariva, L., 2003. Métodos para avaliação da alimentação natural de peixes: o que estamos usando? Interciência, v. 28, (2), 100-104. Heindler, F.M.; Alajmi, F.; Huerlimann, R.; Zeng, C.; Newman, S.J.; Vamvounis, G.; Van Herwerden, L., 2017. Toxic effects of polyethylene terephthalate microparticles and Di(2-ethylhexyl)phthalate on the calanoid copepod, Parvocalanus crassirostris. Ecotoxicology and Environmental Safety, 141, 298- 305. https://doi.org/10.1016/j.ecoenv.2017.03.029. Huchette, S.M.H.; Beveridge, M.C.M.; Baird, D.J.; Ireland, M., 2000. The impacts of grazing by tilapias Oreochromis niloticus L. on periphyton communities growing on / artificial substrate in cages. Aquaculture, v. 186, (1-2), 45-60. https://doi.org/10.1016/S0044-8486(99)00365-8. Inoue, L.A.K.A.; Bezerra, A.C.; Miranda, W.S.; Muniz, A.W.; Boijink, C.L., 2014. Cultivo de tambaqui em gaiolas de baixo volume: efeito da densidade de estocagem na produção de biomassa. Ciência Animal Brasileira, v. 15, (4), 437-443. https://doi.org/10.1590/1089-6891v15i426758. Inyang, A.I.; Sunday, K.E.; Nwankwo, D.I., 2018. Composition of periphyton community on water hyacinth (Eichhornia crassipes): In analysis of environmental characteristics at Ejirin part of Epelagoon in southwestern Nigeria. Journal of Marine Biology, v. 2015, 376986. https://doi. org/10.1155/2015/376986. Keshavanath, P.; Gangadhar, B.; Ramesh, T.J.; Van Dam, A.A.; Beveridge, M.C.M.; Verdegem, M.C.J., 2004. Effects of bamboo substrate and supplemental feeding on growth and production of hybrid red tilapia fingerlings (Oreochromis mossambicus x Oreochromis niloticus). Aquaculture, v. 235, (1-4), 303-314. https://doi.org/10.1016/j. aquaculture.2003.12.017. Keshavanath, P.; Gangadhar, B.; Ramesh, T.J.; Van Rooij, J.M.; Beveridge, M.C.M.; Baird, D.J.; Verdegem, M.C.J.; Van Dam, A.A., 2001. Use of artificial substrates to enhance production of freshwater herbivorous fish in pond culture. Aquaculture Research, v. 32, (3), 189-197. https://doi.org/10.1046/ j.1365-2109.2001.00544.x. Le Cren, E.D., 1951. The length-weight relationship and seasonal cycle in gonad weight and condition in the perch (Perca fluviatilis). Journal of Animal Ecology, v. 20, (2), 201-219. https://doi.org/10.2307/1540. Loures, B.T.R; Ribeiro, R.P.; Vargas, L.; Moreira, H.L.M.; Sussel, F.R., Povh, J.A.; Cavichiolo, F., 2001. Manejo alimentar de alevinos de tilápia do Nilo, Oreochromis niloticus (L.), associado às variáveis físicas, químicas e biológicas do ambiente. Acta Scientiarum Maringá, v. 23, 877-883. https://doi. org/10.4025/actascianimsci.v23i0.2640. Miloloža, M.; Kucic Grgic, D.; Bolanca, T.; Ukic, Š.; Cvetnic, M.; Ocelic Bulatovic, V.; Dionysiou, D.D.; Kušic, H., 2021. Ecotoxicological assessment of microplastics in freshwater sources—a review. Water, v. 13, (1), 56. https://doi. org/10.3390/w13010056. Milstein, A.; Peretz, Y.; Harpaz, H., 2008. Culture of organic tilapia to market size in periphyton-based ponds with reduced feed inputs. Aquaculture Research, v. 40, (1), 55-59. https://doi.org/10.1111/j.1365-2109.2008.02062.x. Murdock, J.N.; Dodds, W.K., 2007. Linking benthic algal biomass to stream substratum topography. Journal of Phycology, v. 43, (3), 449-460. https://doi. org/10.1111/j.1529-8817.2007.00357.x. Neal, E.C.; Patten, B.C.; DePoe, C.E., 1967. Periphyton growth on artificial substrates in a radioactively contaminated lake. Ecology, v. 48, (6), 918-924. https://doi.org/10.2307/1934534 Osório, N.; Cunha, E.R.; Tramonte, R.P.; Mormul, R.P.; Rodrigues, L., 2019. Habitat complexity drives the turnover and nestedness patterns in a periphytic algae community. Limnology, v. 20, 297-307. https://doi.org/10.1007/s10201-019-00578-y. Pérez, G.R., 1992. Fundamentos de limnologia neotropical. Editora da Universidade Antioquia, Medellin, 529 pp. http://doi.org/10.1016/j.aquaculture.2017.08.026 http://doi.org/10.1016/j.aquaculture.2017.08.026 https://doi.org/10.1111/j.1095-8649.1989.tb03300.x https://doi.org/10.1016/b978-0-08-100506-4.00001-5 https://doi.org/10.1016/j.watres.2019.115270 https://doi.org/10.1016/j.watres.2019.115270 https://doi.org/10.20950/1678-2305/bip.2021.47.e622 https://doi.org/10.20950/1678-2305/bip.2021.47.e622 https://doi.org/10.1590/S2175-78602012000200018 https://doi.org/10.1016/j.aquaculture.2017.07.024 https://doi.org/10.1590/S1676-06032010000100009 https://doi.org/10.1590/S1676-06032010000100009 https://doi.org/10.1111/are.14850 https://doi.org/10.1111/are.14850 https://doi.org/10.1016/j.ecoenv.2017.03.029 https://doi.org/10.1016/S0044-8486(99)00365-8 https://doi.org/10.1590/1089-6891v15i426758 https://doi.org/10.1155/2015/376986 https://doi.org/10.1155/2015/376986 https://doi.org/10.1016/j.aquaculture.2003.12.017 https://doi.org/10.1016/j.aquaculture.2003.12.017 https://doi.org/10.1046/j.1365-2109.2001.00544.x https://doi.org/10.1046/j.1365-2109.2001.00544.x https://doi.org/10.2307/1540 https://doi.org/10.4025/actascianimsci.v23i0.2640 https://doi.org/10.4025/actascianimsci.v23i0.2640 https://doi.org/10.3390/w13010056 https://doi.org/10.3390/w13010056 https://doi.org/10.1111/j.1365-2109.2008.02062.x https://doi.org/10.1111/j.1529-8817.2007.00357.x https://doi.org/10.1111/j.1529-8817.2007.00357.x https://doi.org/10.2307/1934534 https://doi.org/10.1007/s10201-019-00578-y Contributions of the periphyton to the growth of Nile tilapia (Oreochromis niloticus) fingerlings on different fixation substrates: an ecological approach 341 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 Putro, S.P.; Sharani, J.; Widowati, A.; Suryono, S., 2020. Biomonitoring of the Application of Monoculture and Integrated Multi-Trophic Aquaculture (IMTA) using macrobenthic structures at Tembelas Island, Kepulauan Riau Province, Indonesia. Journal of Marine Science and Engineering, v. 8, (11), 942. https://doi.org/10.3390/jmse8110942. Ramos, I.P.; Brandão, H.; Zanatta, A.S.; Zica, É.O.P.; Silva, R.J.; Rezende- Ayroza, D.M.; Carvalho, E.D., 2013. Interference of cage fish farm on diet, condition factor and numeric abundance on wild fish in a Neotropical reservoir. Aquaculture, v. 414-415, 56-62. https://doi.org/10.1016/j. aquaculture.2013.07.013. R Core Team, (2003). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Rivera Vasconcelos, F.; Menezes, R.F.; Attayde, L., 2018. Effects of the Nile tilapia (Oreochromis niloticus L.) on the plankton community of a tropical reservoir during and after an algal bloom. Hydrobiologia, v. 817, 393-401. https://doi.org/10.1007/s10750-018-3591-2. Russo, M.R.; Leal, F.C.; Mendes, S.G.F; Souza, E.C.V., 2021. A aquicultura sustentável como alternativa de geração de renda. In: Mauad, J.C.; Mussuri, R.M. (Eds.), Centro de desenvolvimento rural do Itamarati: relatos e vivências. Seriema, Dourados, pp. 221-234. Salazar-Torres, G.; Silva, L.H.S.; Rangel, L.M.; Attayde, J.L.; Huszar, V.L.M., 2016. Cyanobacteria are controlled by omnivorous filter-feeding fish (Nile tilapia) in a tropical eutrophic reservoir. Hydrobiologia, v. 765, 115-129. https://doi.org/10.1007/s10750-015-2406-y. Sarkar, U.K.; Khan, G.E.; Dabas, A.; Pathak, A.K.; Mir, J.I.; Rebello, S.C.; Singh, S.P., 2013. Length weight relationship and condition factor of selected freshwater fish species found in River Ganga, Gomti and Rapti, India. Journal of Environmental Biology, v. 34, (5), 951-956. Sipaúba-Tavares, L.H., 1993. Análise da seletividade alimentar em larvas de tambaqui (Colossoma macropomum) e tambacu (híbrido, pacu Piaractus mesopotamicus e tambaqui Colossoma macropomum) sobre os organismos zooplanctônicos. Acta Limnologica Brasiliensia, v. 6, (1), 114-132. Siqueira, N.S.; Rodrigues, L., 2009. Biomassa perifítica em tanques-rede de criação de tilápia do Nilo Oreochromis niloticus (Linneau, 1758). Boletim do Instituto de Pesca, v. 35, (2), 181-190. Tedeschi, A.C.; Chow-Fraser, P., 2021. Periphytic algal biomass as a bioindicator of phosphorus concentrations in agricultural headwater streams of southern Ontario. Journal of Great Lakes Research, v. 47, (6), 1702-1709. https://doi.org/10.1016/j.jglr.2021.08.018. Tortolero, S.A.R.; Cavero, B.A.S.; Brito, J.G.; Soares, C.C.; Silva Junior, J.L.; Barbosa, H.T.B.; Gangadhar, B.; Keshavanath, P., 2015. Periphyton-based polyculture of jaraqui, Semaprochilodus insignis (Schomburgk, 1841) and tambaqui, Colossoma macropomum (Cuvier, 1816) with feed supplementation. Journal of Aquaculture in the Tropics, v. 30, (3-4), 111-132. Uddin, M.S.; Azim, M.E.; Wahab, M.A.; Verdegem, M.C.J., 2009. Effects of substrate addition and supplemental feeding on plankton composition and production in tilapia (Oreochromis niloticus) and freshwater prawn (Macrobrachium rosenbergii) polyculture. Aquaculture, v. 297, (1-4), 99-105. https://doi.org/10.1016/j.aquaculture.2009.09.016. Uys, W.; Hecht, T., 1985. Evaluation and preparation of an optimal dry feed for the primary nursing of Clarias garipineus larvae (Pisces: Clariidae). Aquaculture, v. 47, (2-3), 173-183. https://doi.org/10.1016/0044- 8486(85)90063-8. Van Dam, A.A.; Beveridge, M.C.M.; Azim, M.E.; Verdegem, M.C.J., 2002. The potential of fish production based on periphyton. Reviews in Fish Biology and Fisheries, v. 12, 1-31. https://doi.org/10.1023/A:1022639805031. Vazzoler, A.E.A.M., 1996. Biologia da reprodução de peixes teleósteos: teoria e prática. Eduem, Maringá, 169 pp. Vercellino, I.S.; Bicudo, D.C., 2006. Sucessão da comunidade de algas perifíticas em reservatório oligotrófico tropical (São Paulo, Brasil): comparação entre período seco e chuvoso. Brazilian Journal of Botany, v. 29, (3), 363-377. https://doi.org/10.1590/S0100-84042006000300004. Wetzel, R.G., 1990. Land-water interfaces: metabolic and limnological regulators. Internationale Vereinigung für theoretische und angewandte Limnologie: Verhandlungen. SIL Proceedings, v. 24, (1), 6-24. https://doi.org/1 0.1080/03680770.1989.11898687. https://doi.org/10.3390/jmse8110942 https://doi.org/10.1016/j.aquaculture.2013.07.013 https://doi.org/10.1016/j.aquaculture.2013.07.013 https://doi.org/10.1007/s10750-018-3591-2 https://doi.org/10.1007/s10750-015-2406-y https://doi.org/10.1016/j.jglr.2021.08.018 https://doi.org/10.1016/j.aquaculture.2009.09.016 https://doi.org/10.1016/0044-8486(85)90063-8 https://doi.org/10.1016/0044-8486(85)90063-8 https://doi.org/10.1023/A https://doi.org/10.1590/S0100-84042006000300004 https://doi.org/10.1080/03680770.1989.11898687 https://doi.org/10.1080/03680770.1989.11898687 Rosa, Y. P. S. et al 342 RBCIAMB | v.57 | n.2 | June 2022 | 333-342 - ISSN 2176-9478 Appendix 1 – Absence and presence of the taxa found in the three fixation substrates throughout the 30 days of the experiment. PET GEOMEMBRANE BAMBOO Arcella x Synhymeniida X x Peritrichida x x x Peniculida x x Ictio sp x Suctoria x x x Chytridiomycetes x x x Bdelloidea x x x Eurotatoria x x Eustigmatales x x Volvocales x Ulotrichales x x x Eudorina x Chroococales x x x Oscillatoriales x x Chlorococcales x x Coelastrum x x x Desmodesmus x x Monoraphidium x x Scenedesmus x x x Eutetramorus x x x Pennales x x Oocystis sp x x Selenastrum sp x x Mycrocistis sp x x Anabaena sp x x Bulbochaete sp x Rizoclonium sp x Closterium sp x Oedogonium sp x Nauplius x Resistance eggs x x x Diptera larvae x