Chronic toxicity of the synthetic hormone 17alpha-ethinylestradiol to Chironomus tentans and Hyalella azteca

The chronic toxicity of the synthetic hormone 17alpha-ethinylestradiol (EE2) was investigated in two benthic invertebrates, the midge Chironomus tentans and the freshwater amphipod Hyalella azteca, in life-cycle water-only assays. In C. tentans, a 50% decrease in emergence was observed at a concentration of 1.5 mg/L; emergence was a more sensitive endpoint than survival, growth, or biomass. Reproduction was not significantly affected by EE2 exposure until a concentration of 3.1 mg/L, where emergence, and therefore reproduction, did not occur. In contrast, reproduction was the most sensitive endpoint in H. azteca (50% decrease in reproduction observed at a concentration of 0.36 mg/L). The sensitivity of the F1 generation to EE2 was also investigated with H. azteca, but was not different from the F0 generation. The data from the present study were combined with those from previous 10-d toxicity assays, to derive acute to chronic toxicity ratios (ACRs) for EE2. The ACRs calculated for EE2 were 13 for C. tentans and 16 for H. azteca, indicating that the application factors currently used in ecological risk assessment for the derivation of chronic toxicity are protective and conservative for these organisms. The results of the present study suggest that chronic toxicity was not mediated by disruption of endocrine pathways. Using a hazard quotient approach, the risk associated with sublethal exposure to EE2 was <1 for H. azteca and C. tentans, indicating that adverse effects are not expected, and that environmental exposure to EE2 likely poses a low risk to benthic invertebrates.     

A field assessment of long-term laboratory sediment toxicity tests with the amphipod Hyalella azteca

Response of the amphipod Hyalella azteca exposed to contaminated sediments for 10 to 42 d in laboratory toxicity tests was compared to responses observed in controlled three-month invertebrate colonization exposures conducted in a pond. Sediments evaluated included a sediment spiked with dichlorodiphenyldichloroethane (DDD) or dilutions of a field sediment collected from the Grand Calumet River (GCR) in Indiana (USA) (contaminated with organic compounds and metals). Consistent effects were observed at the highest exposure concentrations (400 microg DDD/goc [DDD concentrations normalized to grams of organic carbon (goc) in sediment] or 4% GCR sediment) on survival, length, and reproduction of amphipods in the laboratory and on abundance of invertebrates colonizing sediments in the field. Effect concentrations for DDD observed for 10-d length and 42-d reproduction of amphipods (e.g., chronic value [ChV] of 66 microg DDD/goc and 25% inhibition concentration [IC25] of 68 microg DDD/ goc for reproduction) were similar to the lowest effect concentrations for DDD measured on invertebrates colonizing sediment the field. Effect concentrations for GCR sediment on 28-d survival and length and 42-d reproduction and length of amphipods (i.e., ChVs of 0.20-0.66% GCR sediment) provided more conservative effect concentrations compared to 10-d survival or length of amphipods in the laboratory or the response of invertebrates colonizing sediment in the field (e.g., ChVs of 2.2% GCR sediment). Results of this study indicate that use of chronic laboratory toxicity tests with H. azteca and benthic colonization studies should be used to provide conservative estimates of impacts on benthic communities exposed to contaminated sediments. Bioaccumulation of DDD by oligochaetes colonizing the DDD-spiked sediment was similar to results of laboratory sediment tests previously conducted with the oligochaete Lumbriculus variegates, confirming that laboratory exposures can be used to estimate bioaccumulation by oligochaetes exposed in the field.     

Response spectrum of pentachlorobenzene and fluoranthene for Chironomus tentans and Hyalella azteca

The whole-body residues of pentachlorobenzene (PCBz) and fluoranthene (FLU) in Hyalella azteca and Chironomus tentans were determined for a variety of chronic sublethal effects. The endpoints evaluated for H. azteca included 28-d growth and survival and 42-d growth, survival, and reproduction. Adverse effects to C. tentans also were determined at multiple endpoints including 10-d growth, cumulative pupation and emergence, and reproduction. The lowest-observed-effect residue (LOER) based on whole-body residues associated with growth was consistent between compounds and species tested with concentrations ranging from 0.17 to 0.33 micromol/g. For H. azteca, the most sensitive endpoints were growth at 0.23 micromol/g and reproduction at 0.11 micromol/g for PCBz and FLU, respectively. For C. tentans, the most sensitive endpoints were emergence, development and reproduction at 0.02 micromol/g, and development and reproduction at 0.15 micromol/g for PCBz and FLU, respectively. Compared to residues associated with acute lethality, the most sensitive sublethal endpoints were approximately 4 and 60 times lower for PCBz and FLU, respectively. The relative consistency of the sublethal endpoints suggests that body residues can be a valuable tool to evaluate bioaccumulation data as part of a risk assessment to predict adverse effects to biota.     

Lethal and sublethal effects of low dissolved oxygen condition on two aquatic invertebrates, Chironomus tentans and Hyalella azteca

Low dissolved oxygen (DO) conditions occur frequently during sediment toxicity testing, with potentially adverse effects on test organisms. The present study addressed the current lack of good information regarding low DO thresholds for toxicity tests using two common test species, juvenile Hyalella azteca and Chironomus tentans larvae. Results indicated that H. azteca was less tolerant of hypoxia than C. tentans. The 10-d highest- and no-observed-effect concentrations (HOEC and NOEC, respectively) for H. azteca were 1.2 +/- 0.1 and 2.9 +/- 0.1 mg/L DO, respectively. The 10-d NOEC for C. tentans was 1.2 +/- 0.1 mg/L DO, the lowest test concentration. Mortality was the predominant response of H. azteca to low DO exposure, with changes in growth and positioning behavior only evident at lethal DO concentrations. Although exposure to 1.2 +/- 0.1 mg/L DO for 10 d did not affect C. tentans survival or growth, significant behavioral changes were evident at 2.0 +/- 0.1 mg/L DO or less. Overall, the present results indicate that the North American guidelines for low DO thresholds during 10-d toxicity tests seem reasonable for juvenile H. azteca. However, the Environment Canada Guideline (3.4 mg/L DO at 23 degrees C) may be considered to be conservative for 10-d toxicity testing with C. tentans if only short-term effects on survival and growth are considered.     

Sediment Toxicity Evaluation for Hexachlorobenzene: Spiked Sediment Tests with Leptocheirus plumulosus, Hyalella azteca, and Chironomus tentans

Hexachlorobenzene (HCB) is a hydrophobic organic chemical that has shown a lack of toxicity in aquatic tests at concentrations up to and exceeding the solubility limit. The equilibrium partitioning approach to deriving sediment quality benchmarks, which assumes that toxicity can be predicted based on contaminant concentrations in interstitial water, predicts that HCB will not produce direct toxicity to benthic invertebrates as a sediment contaminant. However, the potential for toxicity due to direct exposure to sediment-adsorbed HCB has not been thoroughly established. This study evaluated the survival and growth of the estuarine amphipod Leptocheirus plumulosus, the freshwater amphipod Hyalella azteca, and the midge Chironomus tentans (freshwater) following 10-day exposure to sediment spiked with a range of HCB concentrations. H. azteca was tested under both freshwater and estuarine (10 ppt salinity) conditions. No significant toxicity was observed for any test species at the highest test concentration (60 mg/kg normalized to 1% organic carbon). Minimum detectable differences were less than or equal to 20% for three of eight test endpoints. The observed results add to the available weight of evidence indicating a limited potential for HCB-related sediment toxicity to benthic invertebrates. Received: 23 February 1998/Accepted: 8 June 1998     

Toxicity of uranium, molybdenum, nickel, and arsenic to Hyalella azteca and Chironomus dilutus in water-only and spiked-sediment toxicity tests

A series of laboratory spiked-sediment toxicity tests with the amphipod Hyalella azteca and the midge Chironomus dilutus were undertaken to determine acute and chronic toxicity thresholds for uranium (U), molybdenum (Mo), nickel (Ni), and arsenic (As) based on both whole-sediment (total) and pore water exposure concentrations. Water-only toxicity data were also generated from separate experiments to determine the toxicities of these metals/metalloids under our test conditions and to help evaluate the hypothesis that pore water metal concentrations are better correlated with sediment toxicity to benthic organisms than whole-sediment metal concentrations. The relative toxicity of the four elements tested differed depending on which test species was used and whether whole-sediment or pore water metal concentrations were correlated with effects. Based on measured whole-sediment concentrations, Ni and As were the two most acutely toxic elements to H. azteca with 10-d LC50s of 521 and 532 mg/kg d.w., respectively. Measured pore water concentrations indicated that U and Ni were the two most acutely toxic elements, with 10-d LC50s to H. azteca of 2.15 and 2.05 mg/L, respectively. Based on pore water metal concentrations, the no-observed-effect concentrations (NOECs) for growth were (H. azteca and C. dilutus, respectively) 0.67 and 0.21 mg/L for U, <0.37 and 0.60 mg/L for Ni, and 16.43 and <0.42 mg/L for As. Pore-water lowest-observed-effect concentrations (LOECs) for growth were (H. azteca and C. dilutus, respectively) 2.99 and 0.48 mg/L for U, 0.37 and 2.33 mg/L for Ni, and 58.99 and 0.42 mg/L for As. For U and Ni, results from 96-h water-only acute toxicity tests correlated well with pore water metal concentrations in acutely toxic metal-spiked sediment. This was not true for As where metalloid concentrations in overlying water (diffusion from sediment) may have contributed to toxicity. The lowest whole-sediment LOEC reported here for As was 6.6- and 4-fold higher than the Canadian Council of Ministers of the Environment interim sediment quality guideline and the Canadian Nuclear Safety Commission (CNSC) lowest effect level (LEL), respectively. The lowest whole-sediment LOECs reported here for Ni, U and Mo were 4-, 17.5-, and >260-fold higher, respectively, than the CNSC LELs for these metals/metalloids. Data on pore water metal concentrations in toxic sediment would be a useful addition to future Guidelines documents.     

Time-dependent toxicity of dichlorodiphenyldichloroethylene to Hyalella azteca

Temporal effects on body residues of dichlorodiphenyldichloroethylene (DDE) associated with mortality in the freshwater amphipod Hyalella azteca were evaluated. Toxicokinetics and body residues were determined from water-only exposures that varied from 4 to 28 d, and DDE concentrations ranging from 0.0013 to 0.045 micromol L(-1). Uptake and elimination parameters were not affected significantly by the various temporal and concentration treatments. Uptake rate coefficients ranged from 134.3 to 586.7 ml g(-1) h(-1), and elimination rate coefficients ranged from 0.0011 to 0.0249 h(-1). Toxicity metric values included body residue for 50% mortality at a fixed sample time (LR50) and mean lethal residue to produce 50% mortality from individual exposure concentrations (MLR50) for live organisms and dead organisms. A twofold increase occurred in the MLR50 values calculated using live organisms compared to MLR50 values using dead organisms. Toxicity and kinetic data were fit to a damage assessment model that allows for the time course for toxicokinetics and damage repair, demonstrating the time-dependence of body residues to toxicity. The DDE appeared to act through a nonpolar narcosis mode of action for both acute and chronic mortality in H. azteca. Furthermore, the temporal trend in the toxic response using body residue as the dose metric is steep and found to be similar to another chlorinated hydrocarbon, pentachlorobenzene, but was more potent than that found for polycyclic aromatic hydrocarbons (PAHs).     

Chemical availability and sediment toxicity of pyrethroid insecticides to Hyalella azteca: application to field sediment with unexpectedly low toxicity

Tenax extraction is a simple, inexpensive approach to estimate the bioavailability of hydrophobic organic contaminants from sediment. In the present study, a single-point Tenax extraction was evaluated regarding its correlation with the acute toxicity to Hyalella azteca using field-collected sediments in California, USA. Pyrethroids were believed to be the primary contributor to the observed toxicity, and a significant correlation existed between the expected toxicity (given pyrethroid concentrations) and the mortality at most sampling sites. A small subset of sites, however, showed unexpectedly low toxicity to H. azteca despite high concentrations of pyrethroids. These samples were evaluated by Tenax extraction with the expectation that this procedure, which qualifies bioavailable instead of total pyrethroid concentration in sediment, would better explain the anomalously low toxicity. The term bioavailable toxic unit was proposed to link sediment toxicity with chemical availability, and the toxicity in the 17 selected sediments was better explained using Tenax extraction. The r2 value of the regression between sediment toxicity and toxic unit for the 17 sediments increased from 0.24 to 0.60 when the Tenax-extractable concentration was used in place of the total concentration. Results also showed that adsorption to sand particles might play a controlling role in pyrethroid bioavailability and, in turn, sediment toxicity to benthic invertebrates.     

Toxicokinetics of sediment-sorbed benzo[a]pyrene and hexachlorobiphenyl using the freshwater invertebrates Hyalella azteca,Chironomus tentans,and Lumbriculus variegatus

This study investigated the effect of long-term sediment aging on the toxicokinetics of benzo[a]pyrene (BaP) and hexachlorobiphenyl (HCBP) using three freshwater benthic invertebrates. Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus were exposed to BaP- and HCBP-spiked sediments that were aged for 7 d or 1.5 years. The toxicokinetics of the two compounds were determined for each test organism using a two-compartment model. The modeling of BaP was more complex because biotransformation was included within the model. The results of this study showed that the HCBP uptake clearance rates (k(s)) for each species were generally an order of magnitude greater than those determined for BaP and this difference was most likely due to preferential and rapid binding of BaP to sediment particles. Overall, the bioavailability of HCBP in spiked sediments tended to decrease with duration of aging, based on k(s) values and bioaccumulation factors (BAFs). However, the decreases in bioavailability appear to be species specific. Benzo[a]pyrene did not decline in bioavailability for the species tested because it may resist movement into the micropores of the sediment due to its large size. In addition to the bioassays, this article outlines a method for toxicokinetic modeling of biotransformed compounds and methods for statistical comparisons of kinetic parameters (i.e., k(s), k(d)...) and BAF values.     

The toxicity of fluoranthene to Hyalella azteca in sediment and water-only exposures under varying light spectra

In the US Environmental Protection Agency methods for sediment toxicity testing, the light regimen is specified as a 16:8 light dark cycle with 500-1000 lx. The potential for photoinduced toxic effects from this requirement is evaluated. Hyalella azteca were exposed to fluoranthene in both water only and sediment to examine the impact of light spectra on the toxicity of fluoranthene. The light sources included gold fluorescent light (lambda > 500 nm), cool white fluorescent light, and UV-enhanced fluorescent light. Toxicity was determined as mortality after 10 days of exposure. The extent of mortality was determined both as LC(50) and LR(50) (median lethal body residue). In water-only exposures, the toxicity of fluoranthene was greatest under the UV-enhanced spectra, followed by fluorescent light, and least toxic under the gold light. Both the LC(50) and LR(50) values exhibited the same pattern. The toxicity under gold light gave an LR(50) of 0.81 mmol kg(-1) (0.82-0.79, 95% CI) similar to values expected for the acute toxicity of nonpolar narcotic (anesthetic) compounds. The LR(50) values under the other two light sources were substantially lower, 4 and 58 times lower for the fluorescent and UV-enhanced exposures, respectively. In sediment, toxicity was not significantly affected by the light source. Toxicity occurred only when the body residue concentration approached that of the LR(50) under gold light from the water-only exposures. Thus, H. azteca were significantly protected from the light by burrowing into the sediment.     

Evaluation of the biotic ligand model to predict long-term toxicity of nickel to Hyalella azteca

Three models were developed and evaluated for their ability to predict long-term bioaccumulation of nickel (Ni) and its toxicity to Hyalella azteca using data from 28-d toxicity tests. One of the models was based on competitive action of Ni with Ca and H (the biotic ligand model; BLM), and the other two models included expressions for the potential noncompetitive action of calcium on the ligand (i.e., acclimation), in addition to, or instead of, its competitive action (not accounted for in the BLM). Each model was able to predict lethal accumulation 50 (accumulation at 50% mortality; LA50s) within a factor of 2 of the corresponding observed LA50. The mean predicted LA50 from all three models was within 13% of the observed mean LA50 of 0.90 μmol/g (dry weight). The median lethal concentrations (LC50s) predicted by the three models were similar and were within a factor of 2 of the observed LC50s for 11 of 13 tests, providing encouragement for further development of a long-term Ni BLM. The similar performance of models based on competitive or noncompetitive action may reflect limitations in the data set or may suggest that effects of calcium on the ligand (L(T)) were insufficient to hamper the functionality of the competitive model or that the LA50/L(T) ratio, rather than the LA50 and L(T), is constant.     

Modeling chronic dietary cadmium bioaccumulation and toxicity from periphyton to Hyalella azteca

A chronic (28-d) Cd saturation bioaccumulation model was developed to quantify the Cd contribution from a natural periphyton diet to Cd in the freshwater amphipod Hyalella azteca. Bioaccumulation was then linked to chronic toxic effects. Juvenile H. azteca were exposed to treatments of Cd in water (3.13-100 nmol/L nominal) and food (389-26,300 nmol/g ash-free dry mass). Cadmium bioaccumulation, survival, and growth were recorded. Dietary Cd was estimated to contribute 21 to 31, 59 to 94, and 40 to 55% to bioaccumulated Cd in H. azteca exposed to treatments of Cd primarily in water, food, and food?+?water, respectively. Survival as a function of Cd lethal body concentration (679 nmol/g; 95% confidence limits, 617-747) was the most robust endpoint. Body concentration integrated all exposure routes. Based on the lethal body concentration, dietary Cd was predicted to contribute markedly (26-90%) to Cd in H. azteca. Cadmium concentration and food nutritional quality (biomass, chlorophyll a, total lipid, fatty acids, total protein) had no effect on H. azteca nutritional quality (total lipid, fatty acids, total protein) but did influence H. azteca dry weight. This research highlighted the importance of including a dietary component when modeling chronic effects of Cd and when refining endpoints for use in ecological risk assessment and water quality guidelines.     

Toxicity of silver in water and sediment to the freshwater amphipod Hyalella azteca

Hyalella azteca was exposed to Ag as AgNO3 over a 10-d period in water and two lake sediments that were selected on the basis of their differences in metal-binding properties. The median lethal concentrations (LC50s) for waterborne exposures were 5.4 and 4.9 microg/L for total and dissolved Ag, respectively. In the sediment containing a lesser quantity of total Ag-binding ligands (i.e., Bond Lake, Douglas County, WI, USA, sediment), an Ag-amended sediment toxicity test resulted in a 10-d LC50 of 0.084 g (i.e., 84,000 microg) Ag/kg dry sediment or 8.6 microg Ag/L of pore water (PW). The no-observed-effect concentration (NOEC) to lowest-observed-effect concentration (LOEC) range was 0.012 to 0.031 g Ag/kg dry sediment, or less than 5.0 to 6.0 microg Ag/L of PW. In the sediment with a greater quantity of total Ag-binding ligands (i.e., West Bearskin Lake, Cook County, MN, USA, sediment), the 10-d LC50 was 2.98 g Ag/kg dry sediment, and the NOEC to LOEC range was 2.15 to 4.31 g Ag/kg dry sediment. Because "dissolved" concentrations of Ag in PW were less than 5.0 microg/L at the critical exposures in the latter test, the bioavailable and toxic form of Ag may have been a weakly associated coprecipitate or colloidal complex with hydrous iron oxides that competitively partitioned to the surface of the gills.     

Time-dependent lethal body residues for the toxicity of pentachlorobenzene to Hyalella azteca

The study examined the temporal response of Hyalella azteca to pentachlorobenzene (PCBZ) in water-only exposures. Toxicity was evaluated by calculating the body residue of PCBZ associated with survival. The concentration of PCBZ in the tissues of H. azteca associated with 50% mortality decreased from 3 to 0.5 micromol/g over the temporal range of 1 to 28 d, respectively. No significant difference was observed in the body residue calculated for 50% mortality when the value was determined using live or dead organisms. Metabolism of PCBZ was not responsible for the temporal response because no detectable PCBZ biotransformation occurred over an exposure period of 10 d. A damage assessment model was used to evaluate the impact and repair of damage by PCBZ on H. azteca. The toxicokinetics were determined so that the temporal toxicity data could be fit to a damage assessment model. The half-life calculated for the elimination of PCBZ averaged approximately 49 h, while the value determined for the half-life of damage repair from the damage assessment model was 33 h.     

Effect of piperonyl butoxide on permethrin toxicity in the amphipod Hyalella azteca

Piperonyl butoxide (PBO) is a synergist of pyrethroid pesticides found in many products for structural pest control, mosquito control, and home and garden uses. Because both PBO and pyrethroid residues potentially co-occur in urban creeks, this study determined if environmental levels of PBO were capable of synergizing pyrethroids in the environment. Three types of toxicity tests were conducted with the amphipod Hyalella azteca to determine the minimum PBO concentration required to increase toxicity of the pyrethroid permethrin: Sediment was spiked with permethrin only; permethrin and overlying water spiked with PBO; and permethrin, PBO, and overlying water spiked with PBO. In tests with PBO added to both water and sediment, PBO concentrations of 2.3 microg/L in water and 12.5 microg/kg in sediment reduced the permethrin median lethal concentration (LC50) nearly 50% to 7.3 mg/kg organic carbon (OC). Higher concentrations of PBO increased permethrin toxicity up to sevenfold. In exposures with PBO in water alone, 11.3 microg/L was required to increase permethrin toxicity. Urban creek sediments from California and Tennessee, USA, had PBO concentrations in the low microg/kg range; only one water sample was above the detection limit of 0.05 microg/L. Wetlands in northern California also were sampled after application of pyrethrins and PBO for mosquito abatement. Sediment and water PBO concentrations within 12 h of abatement spraying peaked at 3.27 microg/kg and 0.08 microg/L, respectively. These results suggest that environmental PBO concentrations rarely, if ever, reach concentrations needed to increase pyrethroid toxicity to sensitive organisms, though available data on environmental levels are very limited, and additional data are needed to assess definitively the risk.     

Exposure-related effects on Cd bioaccumulation explain toxicity of Cd-phenanthrene mixtures in Hyalella azteca

Little is known regarding mixture effects of metals and polynuclear aromatic hydrocarbon (PAHs) under environmentally relevant exposure regimes. Standard U.S. Environmental Protection Agency (U.S. EPA) procedures were applied and extended to test effects of phenanthrene (Phen) on sediment-Cd uptake, aqueous-Cd uptake, and Cd-elimination kinetics in the amphipod Hyalella azteca. In sediment exposures, Phen increased the projected equilibrium-tissue concentration of Cd from 47.2 (36.2-58.3) to 221.1 microg/g ([117.8-324.3], 95% confidence intervals [CI] in parentheses). Although Cd bioaccumulation increased markedly in sediment exposures, dissolved Cd concentrations and physical-chemical parameters indicative of Cd bioavailability were unaffected by Phen. Further, in water-only exposures, Phen had no effect on Cd bioaccumulation or Cd-elimination kinetics. These results indicate that increased Cd bioaccumulation in Cd-Phen mixtures occurred via a sediment-mediated process and was likely a function of increased uptake associated with feeding (i.e., Phen-induced alterations in ingestion and/or digestive processes). Observed increases in H. azteca lethality when exposed to Cd-Phen mixtures in sediment, but not in water-only exposures, likely resulted from increased Cd bioaccumulation rate rather than a true toxicological synergism. Thus, apparent synergisms and antagonisms may result from exposure-mediated effects in sediment that are unrelated to toxicological interactions. Implications of these findings regarding sediment-quality assessment and suggestions for future studies are discussed.     

Acute and chronic toxicity of lead in water and diet to the amphipod Hyalella azteca

We evaluated the influence of waterborne and dietary lead (Pb) exposure on the acute and chronic toxicity of Pb to the amphipod Hyalella azteca. Test solutions were generated by a modified diluter with an extended (24-h) equilibration period. Acute (96-h) toxicity of Pb varied with water hardness in the range of 71 to 275 mg/L as CaCO3, despite similar dissolved Pb concentrations. Acute toxicity was greatest in soft test water, with less than 50% survival at the lowest dissolved Pb concentration (151 microg/L). Survival also was significantly reduced in medium-hardness water but not in hard test water. In chronic (42-d) studies, amphipods were exposed to waterborne Pb and fed either a control diet or a diet equilibrated with waterborne Pb levels. For animals fed the control diet, the median lethal concentration (LC50) for Pb was 24 degrees g/L (as dissolved Pb), and significant reductions in survival occurred at 16 microg/L. Exposure to Pb-treated diets significantly increased toxicity across a wide range of dissolved Pb concentrations, with a LC50 of 16 microg/L and significant reductions in growth and reproduction at 3.5 microg/L. Significant effects on growth and reproduction occurred at dissolved Pb concentrations close to the current U.S. chronic water-quality criterion. Our results suggest that both aqueous- and dietary-exposure pathways contribute significantly to chronic Pb exposure and toxic effects in aquatic biota.