Avainsana-arkisto: Lumbriculus

We tested different application methods of activated carbon for sediment remediation

This blog post is based on our recently accepted publication in Water Research (Vol. 114, p. 104-112; http://dx.doi.org/10.1016/j.watres.2017.02.025). It is available free of charge until 15. April 2017 under this link.

Activated carbon is a sorbent with the capability of strongly binding pretty much any organic substance to its surface (a process called adsorption). Since a large share of pollution in aquatic ecosystems concerns such organic substances (for example PCB’s), it would hence be a suitable sorbent to remediate them. Once a pollutant is adsorbed to the activated carbon, it is bound so strong that it is no longer available to organisms. This includes even the case where they eat the activated carbon particles “coated” with the pollutant. The organisms would just pass it through their digestive system, pooping it out unaltered. So, long story short, the idea is to render pollutants harmless to the environment, rather than having to remove them (which additionally leaves the question where to put the removed pollutant).

Unfortunately it has been shown that activated carbon itself can actually be quite harmful to certain animals. Therefore, it is necessary to not focus solely on developing these novel remediation methods to be as effective as possible, but to ensure that they are also safe to apply in the environment. After all, what does it help us if we treat the pollution in a place, but at the same time wreck its ecosystem?

In the paper this post is based on, we mainly examined several different methods of applying activated carbon to polluted sediments (which is where the major share of pollutants in aquatic ecosystems are). You basically have two general options: the more laborious one of mixing the sorbent into the sediment actively, or the more “crude” way of thin layer capping. In the latter method you just cover the polluted sediment with the activated carbon (see picture 1). In the field that would mean all you need to do is to take a shovel and spread the carbon. So, while we did know that thin layer capping would be the easier method to execute, what we aimed to find out in our tests was how it compares in matters of effectiveness and safety.

Picture 1: The setup of our test vessels (only thin layer cap tests shown). The activated carbon is applied as a thin layer on top of PCB polluted sediment. Underneath, the burrows of the test organism (Lumbriculus variegatus – a worm living in the sediment) are visible.

We simulated the two application methods in the laboratory in test vessels containing sediment from a PCB-polluted site (Lake Kernaalanjärvi, southern Finland). As a test organism we used Lumbriculus variegatus, small worms that burrow through the sediment. The amount of PCB’s that the worms take up from the test sediments told us how well the different treatments work for remediation, while their biological responses (things like their change in body mass) were used as parameters to measure the adverse effects of the sorbent material itself.

Picture 2: A quick graphical overview on the results of our results: Adverse effects can be comparably high, but remediation efficiency (meaning the reduction of the uptake of a pollutant (here: PCB) is best when activated carbon is mixed into the sediment.

The major results published in this paper were both promising and worrying at the same time (picture 2). We found out that both methods are effective in general. Worms living in sediment under a thin layer cap took up ~50% less PCB’s from the sediment than from the untreated, “raw” sediment. When the activated carbon was mixed into the sediment, the uptake of PCB’s was prevented almost completely. So, while thin layer capping is a method that is a lot easier to use (and hence cheaper), it is not quite as effective as mixing the sorbent into the sediment. Nevertheless, one has to also keep in mind, that animals dwelling in the sediment (and the thin layer cap) can mix the activated carbon with the underlying sediment. This process is called bioturbation and it was actually even visible in our laboratory test vessels (picture 1). It’s just a lot slower than mixing sediment and sorbent right away upon application. In addition, mixing via bioturbation of course requires animals to stay on the treated site and not to flee the site in panic when the activated carbon is applied.

And that’s exactly where our more worrying results come in: the adverse effects of the sorbent itself. With both application methods it became quite apparent that the worms did not really like our miniature-scale remediation works. They lost their appetite almost completely, stopped feeding and hence lost a lot of weight. While that may sound like a desirable achievement to some humans, for our worms that could be a serious issue.

A possible explanation for this sudden loss in appetite was found on electron microscope images that we took (picture 3). It looked like the activated carbon had quite some detrimental effect to the worms’ gut walls. Their microvilli, which are responsible for nutrient absorption from the gut content, were damaged severely in most worms exposed to sediment treated with the sorbent – no matter with what application method. The exact mechanism on how activated carbon causes this kind of damage remain obscure; one suggestion for example is mechanical abrasion (the carbon particles are quite sharp), but also the strong sorption capacity of the material might be involved.

Picture 3: Activated carbon can damage the gut walls (specifically the microvilli) of Lumbriculus variegatus. Image as seen through an electron microscope at a 6000x magnification.

One interesting thing we saw was that thin layer capping with activated carbon can have quite a devastating effect on Lumbriculus variegatus. This is not too surprising, since the organisms are exposed to a high dose of pure activated carbon at the sediment-water interface. However, when we mixed the activated carbon with clay before applying (thus creating a thin layer cap that resembles natural sediment that is enriched with the sorbent), the adverse effects were a lot less severe.  This doesn’t mean there were no more adverse effects, but rather that they were at a comparable level to our other tested application method of mixing the activated carbon into the sediment.

Picture 4: The transmission electron microscopy images (which you saw above) in the making. Photo: Inna Nybom.

From the results seen in this study we were able to draw some conclusions and implications for future field applications. To sum up, both methods are effective. What the thin layer capping method lacks in immediate effectiveness, it makes up for with its easier application and lower costs. When it comes to the adverse effects, we showed that neither one of the methods has a significant advantage over the other – if certain precautions, like avoiding to apply pure activated carbon, are made. So when deciding on a method, the important factors are mostly the available budget and equipment. Thin layer capping is a better option for sediment remediation in cases where special equipment required for other methods cannot be brought in easily (remote areas) or simply in cases where funds are limited. However, before deciding whether or not to utilize activated carbon in general (and big scale), we will have to make sure that its own adverse effects to the environment are not worse than the pollution effects!

Lastly – if you check our blog post on the first field trial of activated carbon based sediment remediation in Finland, you will probably spot some of these implications already “in action”!

Text: Sebastian Abel

Pictures: Sebastian Abel, Inna Nybom

Ällömadot ja ihanat kirput – laboranttiharjoittelijan kokemuksia

Yksi vakiotehtäviä harjoittelun aikana oli vesikirppujen huoltaminen. Kirppuja pidetään yllä odottamassa mm. kemikaalien myrkyllisyyden kokeita. Kirpuille annetaan ruoaksi levää, jota myös kasvatetaan samassa kasvatushuoneessa, kasvatushuoneesta löytyy myös ällömatoja (harvasukamatoja) ja chironomus sääskiä.

Harjoittelun mukavimpiin kuuluva asia on ollu kirppujen huolto, ja tykästyin niihin jo alkuvaiheessa. Monet sanovat etteivät ne näytä erityisen mukavilta otuksilta, mutta livenä niiden katselu on todella rauhoittavaa, ja niistä löytyy paljon mielenkiintoista mikroskoopin alla, tai paljain silmin katsellessa.

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Näissä kuvissa näkyy vesikirpun poikasten kasvaminen, koko tämä tapahtuma on vain muutaman päivän sisällä, ja poikaset saattavat hyvissä tapauksissa tehdä oman poikueensa jo noin viikon ikäisinä. Emot kasvattavat munat selässään, ja poikaset kuoriutuvat emon kuoren sisässä. Kun poikaset ovat valmiita, emo avaa kuortaan ja ne syntyvät aikuisen vesikirpun näköisinä, ja kasvavat nopeasti syntymänsä jälkeen.

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Viimeisessä kuvista poikaset ovat jo valmiita syntymään, ja edellisessäkin poikanen on jo melkein aikuisen muotoinen. Näissä molemmissa kuvissa poikaset ovat alle vuorokauden sisällä valmiita syntymään.

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Vesikirput lisääntyvät normaaleissa olosuhteissa suvuttomasti, naaras tuottaa jälkeläisiä ilman koiraiden asiaan puuttumista. Näkyvänä erona koirailla on suussaan pidempi tuntoelin ”sikari”. Tässä kuvassa erottuu naaraan lyhyempi suukappale.

Toinen ympyröity osa on kirpun sydän, joka mielenkiintoisesti sijaitsee niskassa, muutenkin vesikirppujen elinten ja osien sijainnit poikkeavat hyvin paljon ihmisille totutusta, suoli on osin päässä yms.

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Pintamikroskoopin alla kirput erottuvat aivan eri tavalla. Näistä erottuu paremmin muodon pyöreys, mutta mikroskoopin alla katsoessa on silti vaikea nähdä, miten kuori on muodostunut, kuori on kaksiosainen, ja kokonaisuudessaan kirppu muistuttaa lähinnä simpukkaan pukeutunutta merihevosta. Sisäosat ovat suurelta osin erilliset kuoreen nähden, ja kirput kasvaessaan vaihtavat kuorta.

Tein harjoittelun aikana myös oman kokeilun. ->toksisuuskokeissa käytetään myös harvasukamatoja, joiden kokeen onnistumisen takia täytyy aloittaa syömään sedimenttiä samoihin aikoihin. Se onnistuu leikkaamalla mato puoliksi pari viikkoa ennen kokeen aloittamista, jolloin madot kasvattavat uuden pään. Jakautuminen on harvasukamadoille luonnollinen tapa lisääntyä, ja leikkaaminen käynnistää tapahtuman samalla tavalla kuin luonnollinen katkeaminen. Joten, halusin tietää selviääkö mato jos sen leikkaa useampaan osaan, kokeissa on käytetty vain joko pää- tai häntäpuolta.

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Leikkasin kymmenen matoa siis viiteen osaan. Madon palat elivät muutaman viikon samanlaisissa olosuhteissa kuin muutkin kasvatushuoneen madot. Lopulta kun laskin elävien matojen määrän, olin kieltämättä vähän yllättynyt. Vaikkeivat madot olleet lisääntyneet, niitä ei myöskään ollut kuollut. Kaikista purkeista oli kuollut vain kaksi matoa. Keskimmäisestä osasta kasvaneet olivat kaikkein terveimmän oloisia, ja hännänpään palat olivat vain juuri ja juuri kasvattaneet uuden pään.

Ällömadot on jänniä mut silti ällöjä. Mut kirput on jänniä ja hitsin ihanoita.

Teksti ja kuvat: Risto Pöhö

Nice handwork and beautiful lab ware – building up the experiment and the first sampling

In my previous post, I told about preparations before an experiment can be started. Some more preparations  were still needed while the worms were creating new heads. Exposure sediments should be prepared – spiked, as we call it. Necessary amount of fullerenes to be added to the sediment was calculated after determining concentration of the fullerene suspension; concentration measurements are pretty beautiful, because of the purple color of fullerenes in the measuring solution.  Spiking is done by “a home-made spiking machine”, which means a metal blade stirred by a drill: it provides forceful mixing of chemical to sediment. Also, artificial freshwater for exposure jars was prepared.

Spiking the sediment with fullerene nanoparticles.
Preparing everything for the experiment. In the middle, spiking the sediment with fullerene nanoparticles.

Everything was finally ready for building up the experiment: spiked sediment, size-synchronized worms and artificial freshwater. The next step was building up the exposure jars with an aeration system. At first, the sediment was placed on the bottom and then artificial freshwater was carefully poured above the sediment. No matter how you pour the water, you always have a blended mix which has to let settle for one or two days before aeration can be started and the worms added. The next step is to let the exposure go on and maintain pH and oxygen content at suitable level for the worms.

Microcosmos with Lumbriculus variegatus, the tubes are for aeriation.
Microcosmos with Lumbriculus variegatus, the tubes are for aeriation. On the right, the worms are in their typical feeding position.

After 7 days it was time to collect the first worm samples, which means whole-day handwork. And how to carry that out? The exposure sediment was poured to a sieve and then carefully seek and pick up every worm using a dentist tool.

In the end, you must find your worms. Sieving is a handy method for that.
In the end, you must find your worms. Sieving is a handy method for that.

The worms are put to clean water to empty their guts before they are ready to be weighed in hand-made –how else 😉 – tiny foil cups. After recording wet weights, the worms are either dried or transferred to a freezer waiting for fullerene analysis.

Our laboratorian trainee Risto Pöhö weighing the worms at the end of the experiment. Note the handy tool for making weighing cups.
Our laboratorian trainee Risto Pöhö weighing the worms at the end of the experiment. Note the handy tool for making weighing cups.

Text by Kukka Pakarinen

Pictures by Kaisa Figueiredo, Risto Pöhö, and Kukka Pakarinen

Here we go again – Many steps to an experiment on black worms

After a year as a teacher I came back to research in aquatic ecotoxicology. I’ll test a method to analyze fullerene nanoparticles in separated tissue fractions of black worms. Simply, I’ll expose the worms to fullerenes, collect organisms, fractionate their tissues, and then measure fullerene concentrations in each tissue fraction. But starting a new experiment requires a plenty of preparations in the lab before actual test can be started. Here I tell what is going on during the first two weeks.

I would need a test sediment treated with fullerenes. For the test sediment, I would need fullerenes suspended to water to be added to a natural sediment from Lake Höytiäinen. Luckily, we already had the sediment in our lab… if we didn’t have, I would have to wait for winter to go to the field and collect it through ice… I would also need my test species, black worms, synchronized to similar physiological condition.

Sediment sampling. Pictures by Kristiina Väänänen and Jarkko Akkanen
Sediment sampling during winter time.

As a very first job, I prepared artificial freshwater, which means a lab-made model of fresh water corresponding “average Finnish freshwater” with its hardness. Then, I used that water to suspend fullerenes. Making fullerene suspension takes time: fullerene powder must be vigorously mixed with water for two weeks before it can be used in the experiment. This mixing process must be done because fullerenes are not soluble in water, but they turn to water-stabile form via water flows and mixing. And when thinking about fullerenes’ fate in natural waters, they can enter to the environment e.g. in waste waters. Thus, water suspension is their first step to bottom sediments. Read more about fullerenes’ environmental fate here: http://onlinelibrary.wiley.com/doi/10.1002/etc.2175/full

Fullerene suspension, picture by Kukka Pakarinen
Fullerene suspension.

Black worms are sediment-dwelling benthic worms. They have important ecological roles in aquatic ecosystems as a food source for fish and as decomposers of sediment material. They can be exposed to fullerenes via wasted sediments. In this experiment I’ll need size-synchronized worms, as some other researchers in our group. That’s why we organized “a worm cutting day” to synchronize more than thousand worms. It means that four of us sat a day in the culture room picking worms from their aquariums to petri dishes, and then separating their head parts and tail parts by a surgeon knife: the head parts grow new tails and tail parts grow new heads. How to identify which part is which? Color of the head is a bit black and thicker whereas the tail is red and thinner. Then, we’ll wait for couple of weeks to let the worms create these new parts. Finally, we’ll get test worms with same size and condition. Dividing to heads and tails is also a normal way to reproduce for the black worms. Read more about fullerene-exposed black worms here: http://www.sciencedirect.com/science/article/pii/S0269749111003848

Worm cutting day
Worm cutting day
Head part, tail part and cutting
Head part, tail part and cutting

While fullerene suspension and the worms are underway, I can do some other preparations. Sediment dry weight must be known to adjust volume of fullerene suspension. Preparations for the dry weight could be favorite job for kids: wet sediment is homogenized with a perforated piston before samples are placed to weighing jars and dried.

Mixing and weighing the sediments
Mixing and weighing the sediments

Next week it’s time to measure fullerene concentration in the suspension, add fullerenes to sediment and let them stay to equilibrate before the experiment.

Text by Kukka Pakarinen

Pictures by Jarkko Akkanen, Kristiina Väänänen, Kukka Pakarinen, and Risto Pöhö