Bioconcentration and depuration of endosulfan sulfate in mosquito fish (Gambusia affinis)

Endosulfan is an insecticide which has been widely used in agriculture. The technical grade material consists of two isomers (alpha and beta). Under natural environmental conditions, endosulfan is metabolized through oxidation and the main metabolite in the environment is endosulfan sulfate. Most ecotoxicology research has been conducted with technical grade endosulfan to determine effects on non-target aquatic organisms. Little data on the effects of endosulfan sulfate on aquatic organisms are available in the literature. This study characterizes endosulfan sulfate bioconcentration and depuration in mosquito fish (Gambusia affinis). During the study, G. affinis was exposed to an environmentally relevant endosulfan sulfate concentration of 0.25 μg L⁻¹ for 5 weeks (uptake phase) followed by a 3-week period (depuration phase) in clean water. This study found that G. affinis bioconcentrated endosulfan sulfate. During the exposure phase, fish tissue concentrations of endosulfan sulfate increased with time up to 730 μg kg⁻¹ dw or 215 μg kg⁻¹ ww. The bioconcentration data followed Michaelis-Menten kinetics better than the one-compartment first order kinetics (1-CFOK). Using these models, the bioconcentration factors for endosulfan sulfate-exposed G. affinis were from 687 to 888  L kg⁻¹ in wet weight or 2263 to 2936 L kg⁻¹ in dry weight. During the depuration phase, endosulfan sulfate concentrations in tissue significantly decreased and the data followed first order kinetics. The half-life of endosulfan sulfate in G. affinis was about 9 d. There was no significant difference in standard length or weight between control and exposed fish. The growth data followed the von Bertalanffy growth model. However, the condition factor of exposed fish increased with time during the exposure phase.     

Challenges in assessing the toxic effects of polycyclic aromatic hydrocarbons to marine organisms: a case study on the acute toxicity of pyrene to the European seabass (Dicentrarchus labrax L.)

The acute toxicity (96 h) of pyrene (PY) to European seabass (Dicentrachus labrax) juveniles assessed in a semi-static bioassay (SSB) with medium renewal at each 12 h, and in a static bioassay (SB) without medium renewal was compared in laboratorial conditions (water PY concentrations: 0.07-10 mg L⁻¹). Main findings in the SSB that assessed mainly the toxicity of PY and its metabolites were: increased levels of bile PY metabolites in good agreement with the profile of lipid peroxidation levels (LPO) in exposed fish relating PY exposure and oxidative damage; increased levels of PY-type compounds in the brain indicating their ability to cross the blood-brain barrier; increased levels of these substances in liver and muscle which are edible tissues for humans thus raising concern on potential adverse effects on consumers of fish from PY contaminated areas; a significant inhibition of glutathione S-transferase activity suggesting its involvement in PY detoxication as toxicant scavenger; finally, an almost complete impairment of the swimming velocity at all the PY concentrations linking sub-individual to higher population level effects. In the SB, where the overall toxicity of PY, its metabolites and environmental degradation products was evaluated, 19% and 79% of PY decay in test media was found at 12 and 96 h, respectively. In general, the effects were similar to those of SSB but with significant effects being induced at higher PY concentrations indicating that the parental compound is more toxic than its environmental degradation products. The other main differences relatively to the SSB were: increased levels of PY-type substances in the liver suggesting more accumulation in this organ. Therefore, these findings highlight the need of carefully considering experimental design options when assessing the toxicity of readily degradable substances to marine fish, and stress the importance of taking into consideration the toxicity of environmental degradation products in addition to toxic effects of the parental substance and its metabolites for marine ecological risk assessment.     

Rapid-behaviour responses as a reliable indicator of estrogenic chemical toxicity in zebrafish juveniles

Whereas biochemical and molecular parameters have been well recognised as important “signposts” of individual disturbance to endocrine disrupting chemical’s (EDCs) exposure, behavioural endpoints are yet greatly overlooked as a routine tool in environmental risk assessment of EDCs. However, life histories are intimately associated with numerous inter- and intra-specific interactions, which invariably depend on the performance of effective behaviours. Within fish species, one of the most important factors influencing energy turnover earlier in the development is locomotor activity. This essential trait reflects the organism’s ability to generate and coordinate the metabolic energy required for both reproductive and non-reproductive behaviours. Inappropriate movement responses due to toxic effects of contaminants may ultimately impact important ecological variables.Therefore, in the present study, the swimming bursts of zebrafish juveniles exposed for 40 d to the synthetic estrogen ethinylestradiol (EE₂), tested at environmentally relevant concentrations (nominal concentrations of 0.5, 1 and 2 ng L⁻¹), were investigated in order to address the potential of rapid-behaviour patterns as an effective response indicator of estrogenic endocrine disrupting chemical’s exposure. This synthetic estrogen was selected due to its high prevalence in aquatic ecosystems, ability to mimic natural estrogens and proven record of causing negative effects in fish reproduction. The behavioural responses were compared with established endpoints used in the screening of EE₂ effects at adulthood. Results indicate that zebrafish juveniles’ swimming activity was significantly decreased upon EE₂ exposure. Since reduced locomotion of zebrafish may impact foraging, predator avoidance, drift and transport, and even interfere with social and reproductive behaviours, a fitness decline of wild fish populations can ultimately be hypothesized. Furthermore, behavioural endpoints were found to display higher sensitivity to EE₂ than either vitellogenin gene induction or reproductive parameters determined at adulthood.Overall, the findings of this work not only demonstrate the power of high-throughput behavioural responses, able to act as sensitive early warning signals of EDC exposure, but also highlight the potential of behavioural endpoints in providing a more comprehensive and non-invasive measure of EDC’s exposure.     

Heavy metal (Cd, Cr, Cu, Hg, Pb, Zn) concentrations in seven fish species in relation to fish size and location along the Yangtze River

Purpose

The objective of this paper is to assess the regulation of the accumulation of heavy metals in the aquatic environment and different fish species.

Methods

Water and fish samples were collected from upper to lower reaches of the Yangtze River. The heavy metal (Cd, Cr, Cu, Hg, Pb, Zn) concentrations in the muscle tissue of seven fishes were measured. Additionally, the relationships between heavy metal concentrations in fish tissue and fish size (length and weight), condition factor, water layer distribution, and trophic level were investigated.

Results

Metal concentrations (milligrams per kilogram wet weight) were found to be distributed differently among different fish species. The highest concentrations of Cu (1.22?mg/kg) and Zn (7.55?mg/kg) were measured in Pelteobagrus fulvidraco, the highest concentrations of Cd (0.115?mg/kg) and Hg (0.0304?mg/kg) were measured in Silurus asotus, and the highest concentrations of Pb (0.811?mg/kg) and Cr (0.239?mg/kg) were measured in Carassius auratus and Cyprinus carpio. A positive relationship was found between fish size and metal level in most cases. The variance of the relationships may be the result of differences in habitat, swimming behavior, and metabolic activity. In this study, fishes living in the lower water layer and river bottom had higher metals concentrations than in upper and middle layers. Benthic carnivorous and euryphagous fish had higher metals concentrations than phytoplankton and herbivorous fish. Generally, fish caught from the lower reach had higher metals concentrations than those from the upper reach.

Conclusions

Cadmium and lead concentrations in several fishes exceeded the permissible food consumption limits, this should be considered to be an important warning signal.     

Biochemical biomarkers in Nile tilapia (Oreochromis niloticus) after short-term exposure to diesel oil, pure biodiesel and biodiesel blends

Fossil fuels such as diesel are being gradually replaced by biodiesel, a renewable energy source, cheaper and less polluting. However, little is known about the toxic effects of this new energy source on aquatic organisms. Thus, we evaluated biochemical biomarkers related to oxidative stress in Nile tilapia (Oreochromis niloticus) after two and seven exposure days to diesel and pure biodiesel (B100) and blends B5 and B20 at concentrations of 0.01 and 0.1 mL L⁻¹. The hepatic ethoxyresorufin-O-deethylase activity was highly induced in all groups, except for those animals exposed to B100. There was an increase in lipid peroxidation in liver and gills in the group exposed to the higher concentration of B5. All treatments caused a significant increase in the levels of 1-hydroxypyrene excreted in the bile after 2 and 7 d, except for those fish exposed to B100. The hepatic glutathione-S-transferase increased after 7 d in animals exposed to the higher concentration of diesel and in the gill of fish exposed to the higher concentration of pure diesel and B5, but decreased for the two tested concentrations of B100. Superoxide dismutase, catalase and glutathione peroxidase also presented significant changes according to the treatments for all groups, including B100. Biodiesel B20 in the conditions tested had fewer adverse effects than diesel and B5 for the Nile tilapia, and can be suggested as a less harmful fuel in substitution to diesel. However, even B100 could activate biochemical responses in fish, at the experimental conditions tested, indicating that this fuel can also represent a risk to the aquatic biota.     

The toxicity of titanium dioxide nanopowder to early life stages of the Japanese medaka (Oryzias latipes)

In this study, fertilized Japanese medaka (Oryzias latipes) embryos were exposed from fertilization to 5 d post-hatch using static non-renewal assays to aqueous suspensions of titanium dioxide nanoparticles (nTiO₂) ranging in nominal concentrations between 0 and 14 μg mL⁻¹. The average size of the nTiO₂ in the stock solution before addition to the test treatments was 87 nm (±14 nm). TiO₂ materials accumulated in a concentration dependent manner on the chorionic filaments of developing medaka embryos with evidence of pericardial edema occurring during embryo development. However, no significant (p > 0.05) increases in mortality relative to control treatments were observed for the nTiO₂ exposed embryos. A concentration dependent increase in cumulative percent hatch was observed at 11 d, indicating that exposure to increasing concentrations of nTiO₂ resulted in the premature hatch of medaka embryos. Post-hatch, a significant proportion of sac fry from the nTiO₂ exposure groups exhibited moribund swimming behavior and these individuals also experienced greater mortality at 15 d post-hatch. Combined, these results demonstrate that exposure to nTiO₂ can impact the development of early life stages of fish.     

Toxicity assessment of a common laundry detergent using the freshwater flagellate Euglena gracilis

Synthetic detergents are among the commonly used chemicals in everyday life. Detergents, reaching aquatic environments through domestic and municipal wastewater, can cause many different effects in aquatic organisms. The present study was aimed at the toxicity evaluation of a commonly used laundry detergent, Ariel, using the freshwater flagellate Euglena gracilis as a biotest organism. Different parameters of the flagellate like motility, swimming velocity, cell shape, gravitactic orientation, photosynthesis and concentration of light harvesting pigments were used as end points for the toxicity assessment. No Observed Effect Concentration (NOEC) and EC₅₀ values were calculated for the end point parameters at four different incubation times, i.e. 0, 6, 24 and 72 h. After 72 h incubation, swimming velocity of the cells was found to be the most sensitive parameter giving NOEC and EC₅₀ values of 10.8 and 34 mg L⁻¹, respectively. After 72 h exposure to the detergent, chlorophyll a and total carotenoids were significantly decreased in cultures treated with Ariel at concentrations of 50 mg L⁻¹ and above while chlorophyll b significantly decreased at concentrations above 750 mg L⁻¹. The maximum inhibitory effect on the quantum yield of photosystem II was observed after 24 h exposure and thereafter a recovery trend was observed. Motility, gravitaxis and cell shape were strongly impaired immediately upon exposure to the detergent, but with increasing exposure time these parameters showed acclimatization to the stress and thus the NOEC values obtained after 72 h were higher than those immediately after exposure.     

Sublethal Effects of Monocrotophos on Locomotor Behavior and Gill Architecture of the Mosquito Fish,Gambusia affinis

Subacute studies of monocrotophos [Dimethyl (E)-1-methyl-2-(methyl-carbamoyl) vinyl phosphate] on mosquito fish, Gambusia affinis, were carried out in vivo for 24 days to assess the locomotor behavior, structural integrity of gill, and targeted enzyme acetylcholinesterase (AChE, EC: 3.1.1.7) interactions. Monocrotophos (MCP) can be rated as moderately toxic to G. affinis, with a median lethal concentration (LC₅₀) of 20.49 ± 2.45 mgL^?1. The fish exposed to sublethal concentration of LC₁₀ (7.74 mgL^?1) were under stress and altered their locomotor behavior, such as distance traveled per unit time (m min^?1) and swimming speed (cm sec^?1) with respect to the length of exposure. Inhibition in the activity of brain AChE and deformities in the primary and secondary lamellae of gill may have resulted in failure of exchange of gases. The maximum inhibition of 95% of AChE activity was observed on days 20 and 24.

Morphological aberrations in the gills were also studied during exposure to the sublethal concentration of monocrotophos for a period ranging from 8 to 24 days. The extent of damage in gill was dependent on the duration of exposure. The findings revealed that inhibition in brain AChE activity and structural alteration in gill were responsible for altering the locomotor behavior of exposed fish.     

Uptake, tissue distribution and metabolism of the insecticide endosulfan in Jenynsia multidentata (Anablepidae, Cyprinodontiformes)

The study reports the accumulation, distribution and metabolism of technical endosulfan in Jenynsia multidentata. Adult females were exposed to acute sublethal concentrations (0.072, 0.288 and 1.4 μg L⁻¹). After 24 h, fish were sacrificed and gills, liver, brain, intestine and muscle were removed. Results show that both isomers of technical-grade endosulfan (α- and β-) are accumulated in fish tissues and biotransformation to endosulfan sulfate occurs at all concentrations tested. Significantly differences in endosulfan accumulation were only found at 1.4 μg L⁻¹ but not between the lowest concentrations. However a similar distribution pattern was observed at all exposure levels where liver, intestine and brain had the highest levels of α-, β-endosulfan and endosulfan sulfate. Moreover, liver and brain showed the highest endosulfan sulfate:α-endosulfan ratios due to high biotransfomation capacity. J. multidentata demonstrated to be a sensitive species under exposure to technical endosulfan and, therefore, could be used to assess aquatic pollution.     

Laboratory Investigation of a New Trommel Model

Abstract

Rotary screens, or trommels, are an important unit operation in material and fuel processing. A computer model has been developed based upon fundamental mechanics. The coefficients and variables employed in the model thus have real physical meaning; adjusting them based upon laboratory data allows the model user to draw conclusions about the behavior of the trommel that can be applied to design and operational changes. The laboratory testing program was specifically designed to test the model described in this paper.

The model proved able to track the laboratory data. Important phenomena that were validated or revealed included the significance of particle layering, changes in bed sliding with rotational velocity, and the pre-eminence of residence time.     

Reactive plume model: Effect of stack exit conditions on gas phase precursors and sulfate formation

A simple form of a carbon-bond smog mechanism has been introduced into a complete set of plume conservation equations. The conservation expressions for mass, momentum, energy and component species have been solved numerically for typical source and neutral or adiabatic ambient conditions to simulate the homogeneous gas phase chemistry for approximately two hours of travel time downwind from the stack exit. The influence of stack exit conditions including the ratios of momentum-to-buoyancy length l_m/l_b, source-to-ambient velocity R and mixing-to-reaction time τ are demonstrated.It was found that homogeneous processes in the plume near-field do not significantly contribute to the overall conversion of SO₂ to SO²⁻₄. In the far-field maximum sulfate formation rates of 2.3% h⁻¹ were predicted for clear summer noon hours, consistent with plume measurements. Variation of stack exit conditions were found to have little effect on the maximum SO₂ conversion rate or the peak OH radical concentration where local HC/NO_x ratios between 16 and 22 were predicted. Parameter changes resulting in greater ambient entrainment rates, however, were found to shift the development of the radical pool closer to the source and to significantly increase the total molar flux of plume sulfate.     

Shoreline development and degradation of coastal fish reproduction habitats

Coastal development has severely affected habitats and biodiversity during the last century, but quantitative estimates of the impacts are usually lacking. We utilize predictive habitat modeling and mapping of human pressures to estimate the cumulative long-term effects of coastal development in relation to fish habitats. Based on aerial photographs since the 1960s, shoreline development rates were estimated in the Stockholm archipelago in the Baltic Sea. By combining shoreline development rates with spatial predictions of fish reproduction habitats, we estimated annual habitat degradation rates for three of the most common coastal fish species, northern pike (Esox lucius), Eurasian perch (Perca fluviatilis) and roach (Rutilus rutilus). The results showed that shoreline constructions were concentrated to the reproduction habitats of these species. The estimated degradation rates, where a degraded habitat was defined as having ≥3 constructions per 100 m shoreline, were on average 0.5 % of available habitats per year and about 1 % in areas close to larger population centers. Approximately 40 % of available habitats were already degraded in 2005. These results provide an example of how many small construction projects over time may have a vast impact on coastal fish populations.

Electronic supplementary material

The online version of this article (doi:10.1007/s13280-014-0522-y) contains supplementary material, which is available to authorized users.     

Use of stable sulfur isotopes to identify sources of sulfate in Rocky Mountain snowpacks

Stable sulfur isotope ratios and major ions in bulk snowpack samples were monitored at a network of 52 high-elevation sites along and near the Continental Divide from 1993 to 1999. This information was collected to better define atmospheric deposition to remote areas of the Rocky Mountains and to help identify the major source regions of sulfate in winter deposition. Average annual δ³⁴S values at individual sites ranged from +4.0 to +8.2‰ and standard deviations ranged from 0.4 to 1.6‰. The chemical composition of all samples was extremely dilute and slightly acidic; average sulfate concentrations ranged from 2.4 to 12.2 μeq l⁻¹ and pH ranged from 4.82 to 5.70. The range of δ³⁴S values measured in this study indicated that snowpack sulfur in the Rocky Mountains is primarily derived from anthropogenic sources. A nearly linear relation between δ³⁴S and latitude was observed for sites in New Mexico, Colorado, and southern Wyoming, which indicates that snowpack sulfate in the southern part of the network was derived from two isotopically distinct source regions. Because the major point sources of SO₂ in the region are coal-fired powerplants, this pattern may reflect variations in the isotopic composition of coals burned by the plants. The geographic pattern in δ³⁴S for sites farther to the north in Wyoming and Montana was much less distinct, perhaps reflecting the paucity of major point sources of SO₂ in the northern part of the network.     

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: Linking biomarkers to behaviour

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC₅₀s of copper and mercury (dissolved throughout metal concentrations) at 96 h were 568 μg L⁻¹ and 62 μg L⁻¹, respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 μg L⁻¹ for copper and 3 μg L⁻¹ for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 μg L⁻¹ for copper and from 3 to 25 μg L⁻¹ for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.     

Synergistic effects caused by atrazine and terbuthylazine on chlorpyrifos toxicity to early-life stages of the zebrafish Danio rerio

This study examined the effects of three widely used pesticides that have been previously detected in aquatic systems neighbouring agricultural fields on the early-life stages of the zebrafish Danio rerio. Tests involving single exposures and binary combinations of the s-triazine herbicides (atrazine and terbuthylazine) and the organophosphate insecticide chlorpyrifos were performed. Several endpoints, such as swimming behaviour, morphological abnormalities and mortality, were studied. In addition, the inhibition of acetylcholinesterase (AChE) activity was investigated in order to evaluate the mode of action and toxicity of chlorpyrifos in the presence of these herbicides. Results indicate that both binary mixtures elicited synergistic responses on the swimming behaviour of zebrafish larvae. Moreover, although the herbicides were not effective inhibitors of the AChE on their own, a synergistic inhibition of the enzyme activity was obtained by exposure to mixtures with chlorpyrifos. We observed a correlation between impairment of swimming behaviour of the larvae and inhibition of AChE activity. This study supports previous studies concerning the risk assessment of mixtures since the toxicity may be underestimated when looking only at the single toxicants and not their mixtures.     

Behavioral changes in fish exposed to phytoestrogens

We investigated the behavioral effects of exposure to waterborne phytoestrogens in male fighting fish, Betta splendens. Adult fish were exposed to a range of concentrations of genistein, equol, beta-sitosterol, and the positive control 17beta-estradiol. The following behaviors were measured: spontaneous swimming activity, latency to respond to a perceived intruder (mirror reflection), intensity of aggressive response toward a perceived intruder, probability of constructing a nest in the presence of a female, and the size of the nest constructed. We found few changes in spontaneous swimming activity, the latency to respond to the mirror, and nest size, and modest changes in the probability of constructing a nest. There were significant decreases, however, in the intensity of aggressive behavior toward the mirror following exposure to several concentrations, including environmentally relevant ones, of 17beta-estradiol, genistein, and equol. This suggests that phytoestrogen contamination has the potential to significantly affect the behavior of free-living fishes.     

Source apportionment of time and size resolved ambient particulate matter measured with a rotating DRUM impactor

Ambient particulate chemical composition data acquired from samples collected using a three-stage Davis Rotating-drum Universal-size-cut Monitoring (DRUM) impactor in Detroit, MI, between February and April 2002 were analyzed through the application of a three-way factor analysis model. PM_2.5 (particulate matter ⩽2.5 μm in aerodynamic diameter) was collected by a DRUM impactor with 3-h time resolution and three size modes (2.5 μm>D_p>1.15 μm, 1.15 μm>D_p>0.34 μm and 0.34 μm>D_p>0.1 μm). A novel three-way factor analysis model was applied to these data where the source profiles are a three-way array of size, composition and source while the contributions are a matrix of sample by source. Nine factors were identified: road salt, industrial (Fe+Zn), cloud processed sulfate, two types of metal works, road dust, local sulfate source, sulfur with dust, and homogeneously formed sulfate. Road salt had high concentrations of Na and Cl. Mixed industrial emissions are characterized by Fe and Zn. The cloud processed sulfate had a high concentration of S in the intermediate size mode. The first metal works represented by Fe in all three size modes and by Zn, Ti, Cu, and Mn. The second included a high concentration of small size particle sulfur with intermediate size Fe, Zn, Al, Si, and Ca. Road dust contained Na, Al, Si, S, K, and Fe in the large size mode. The local and homogeneous sulfate factors show high concentrations of S in the smallest size mode, but different time series behavior in their contributions. Sulfur with dust is characterized by S and a mix of Na, Mg, Al, Si, K, Ca, Ti, and Fe from the medium and large size modes. This study shows that the utilization of time and size resolved DRUM data can assist in the identification of sources and atmospheric processes leading to the observed ambient concentrations.     

Dry deposition,surface production and dynamics of aerosols in the marine boundary layer

A model of downward aerosol panicle flux characterized by dry deposition velocity, V_d, due to Slinn and Slinn (1980) is generalized to the case of nonzero surface concentration (absorbing surface with a surface source). A more general expression for the flux at some reference height is developed which includes V_d and an effective surface source strength, S_i, which is a function of the true surface source strength, S_i, and the particle transport properties below the reference height. The general expression for the surface flux is incorporated into a dynamic mixed layer model of the type developed by Davidson et al. (1983). This three layer model (diffusion sublayer, turbulent surface layer and mixed layer) is applied to an open ocean marine regime where boundary layer advection is ignored. The aerosol concentration in the boundary layer is considered to consist of sea salt particles produced as droplets at the surface and ‘continental’ background aerosols brought into the boundary layer at the top by entrainment and gravitational settling. Estimates of S_i are provided.