Effects of ozonation treatment on benthic fauna

How does the density of aquatic invertebrates change in the river Knivsta during removal of pharmaceuticals?

Ludvig Hagberg

Student

Degree Thesis in Biology 15 ECTS Bachelor’s Level

Report passed: XX June 2016 Supervisor: Tomas Brodin

Effects of ozonation treatment on benthic fauna – How does the density of aquatic invertebrates change in the river Knivsta during removal of pharmaceuticals? Ludvig Hagberg

Abstract The quantity of pharmaceuticals that end up in aquatic and terrestrial ecosystems are increasing due to a global increase in usage and poor removal efficiency of pharmaceuticals in sewage treatment plants (STP). These compounds may cause great effects on both aquatic and terrestrial ecosystems. Pharmaceuticals are biologically active compounds that may affect non-target organisms. This report focused primarily on if and how the density of Chironomidae (nonbiting midges), Isopoda, Trichoptera (caddisfly) and Simuliidae (black fly) changed when sewage water was treated with ozonation in the river Knivsta, south of Uppsala. Invertebrates were sampled using the kick-sampling method at 6 sites downstream a STP both before and during ozonation treatment. Furthermore, the biodiversity of invertebrates before and after treatment was studied. Significant differences between densities of Isopoda and Gastropoda were found when comparing samples collected before and during ozonation. Both groups were more abundant during ozonation treatment than before. No trend could be found for effects on biodiversity. Further studies are needed to determine which effects pharmaceuticals have on non-target organisms and also if pharmaceutical removal techniques such as ozonation may have other effects, not related to the removal of pharmaceuticals, on ecosystems and the wildlife therein.

Table of contents 1. Introduction 1

1.1 Background 1 1.2 Changes in behavior due to pharmaceuticals 2 1.3 Effects on benthic fauna 2 1.4 Problem statements 3

2. Method and materials 3 2.1 Method 3 2.2 Statistical analysis 4

3. Result 5 3.1 Biodiversity 5 3.2 Invertebrate density 5

4. Discussion 7 4.1 Biodiversity 7 4.2 Invertebrate density 8

5. References 10

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1. Introduction 1.1 Background Pharmaceuticals are used globally on a daily basis, and in the last decade more and more pharmaceutical residues have been found in aquatic systems (Daughton and Ternes 1999). This is mainly due to a poor removal efficiency of pharmaceuticals in our sewage treatment plants (STP) (Brodin et al. 2014, Grabicova et al. 2015 and Jonsson et al. 2015). The concentrations of pharmaceuticals in aquatic systems are usually low and therefore hard to detect. Only recently with improved analytical technologies have those compounds been observed in the environment, usually in the ng–μg/L range. The active substances in pharmaceuticals may have a great impact on different ecosystems, both aquatic and terrestrial (Sánchez-Argüello, Fernàndez and Tarazona 2009, Gilroy et al. 2012, Arnold et al. 2013 and Brodin et al. 2014). In total over a hundred different pharmaceutical compounds have been found in the environment and presumably there are many more to be discovered (Guo et al. 2016 and Sumpter 2016). There are more than 4000 pharmaceuticals currently on the market, including antibiotics, antihistamines, analgesics and hormones, both for human and veterinary uses. Those compounds as well as personal care products are biologically active compounds which interact with specific processes in the target organism, but may also pose a threat for non-target organisms, in the environment, that carry similar receptors (Marshall and Williams et al. 2011, Royer 2012, Boxall et al. 2012 and Klaminder et al. 2015). As much as 90% of the active drug substance can still remain active after leaving the target organism (Guler and Ford 2010). The degradation rate, toxicity and the rate at which those pharmaceuticals enter non-target organisms can vary depending on the pH of the environment (Williams et al. 2011). Temperature is also an important factor for the degradation rate of pharmaceuticals. The degradation rate increases with warmer temperature and with an increase in pH (Webster, Mackay and Wania 1998). For lindane, an insecticide used in both agriculture and as a treatment for lice and scabies, the half-live can vary from 75 days at 30◦C up to one hundred years at temperatures close to zero (Webster, Mackay and Wania 1998). Even bigger changes in degradation rate can be found when looking at different pH levels. At pH 3 the half-live of lindane is as long as 175 000 years while at pH levels around 9 only 64 days (Webster, Mackay and Wania 1998). Biological factors like habitat preference, life cycle stage, and reproductive strategies may also affect an organism’s uptake of pharmaceuticals (Williams et al. 2011). Organisms that lives on the bottom of a lake or stream may have a greater uptake of pharmaceuticals from the soil compared to organisms that lives exclusively in vegetation (Rubach et al. 2011). However, a predator which feeds on contaminated prey may be more exposed to pharmaceuticals through food intake in comparison to habitat preference (Rubach et al. 2011). Ozone is a gas which is very unstable. The rate of degradation in water ranges from a few seconds to 30 minutes (Water research center 2014). The molecule is composed of three oxygen atoms and due to its instability it’s degrading to O2 which is a far more stable state. Once this happens there is one free oxygen atom which is highly reactive. The free oxygen atom can oxide iron and other metals in water and turn them into an insoluble metal that are removed by filtration. Ozone can also eliminate organic particles, chemicals and reduce odor in water (Water research center 2014). The ozone also has to be removed from the water because of its reactive properties; this can be done using a helium stream (Rodríguez et al. 2013). Rodríguez et al. (2013) has shown that pharmaceuticals from water may be removed in under 30 minutes with the ozonation technique. According to Jiang and Zhou (2013), ferrate (FeVIO4

2−, Fe (VI)) is a chemical which can remove more than 80% of ciprofloxacin in waste water effluent. It may also remove other

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pharmaceuticals such as ibuprofen and naproxen. The use of ferrate with its dual function as an oxidant and coagulant is therefore a promising technique for removal of pharmaceuticals. This technique is however pH dependent and works best at pH levels around 4 (Jiang and Zhou 2013). 1.2 Changes in behavior due to pharmaceuticals Studies have shown significant behavioral alterations in fish due to pharmaceutical impact. For example, European perch (Perca fluviatilis) become more active and bolder and at the same time less social in the presence of the anxiolytic pharmaceutical oxazepam (Brodin et al. 2013 and Brodin et al. 2014). Another study showed that when hybrid striped bass (Morone saxatillis x Morone chrysops) got exposed to sub-lethal concentrations of fluoxetine, an anti-depressant, their ability to catch prey decreased which can lead to reduced fitness (Gaworecki and Klaine 2008). Gaworecki and Klaine (2008) also suggested that serotonin may be used as a biomarker due to its ability to remain in the organism under a non-exposure period as well. Invertebrates are also affected by pharmaceuticals and other pollutants. Fluoxetine in the environment can alter endocrine functions and serotonin levels among invertebrates (Guler and Ford 2010). Changes in serotonin levels among invertebrates may have many effects like changes in reproduction, swimming behavior, egg laying and feeding behavior (Guler and Ford 2010). De Lange et al. (2009) showed that Gammarus pulex exposed to fluoxetine, carbamazepine or ibuprofen showed a dual response for each pharmaceutical. At low concentrations (1-100ng/L) an increase in ventilation (resting) could be found and higher concentrations (1µg/L–1 mg/L) resulted in increased locomotion. A component called CTAB that are used in antiseptics had a more acute effect on G. pulex. A correlation could be found when comparing CTAB concentration with inactivity for G. pulex. At doses between 10-100mg most individuals died within 2 hours (De Lange et al. 2006). Some pharmaceuticals have a more direct impact on species at even lower concentrations. For example in Pakistan the oriental white-backed vulture nearly got extinct because of high levels of diclofenac in livestock that the vultures feed on (Oaks et al. 2004). Other effects from pharmaceuticals range from changes in insect physiology and behavior to effects on algae or aquatic plant growth (Williams et al. 2011). 1.3 Effects on benthic fauna Studies have shown that sediments in aquatic systems may act as a long term source of pharmaceutical compounds due to higher concentrations of those compounds in the sediment compared to the surrounding water (Gilroy et al. 2012). Benthic organisms exposed to such sediment could then be affected (Gilroy et al. 2012). Due to low concentrations of pharmaceuticals in STP effluent water these compounds have gone undetected in aquatic systems until the last 15 years. Therefore, the knowledge of which ecological effects pharmaceuticals may have in aquatic ecosystems are very limited, however more and more studies are focusing on this subject today. More studies on this subject are needed to get a better understanding of the short- respectively long-term effects of pharmaceuticals in our environment. This study focuses on if and how biodiversity and density of benthic fauna are affected by pharmaceuticals in water. More specifically it tests if the density of benthic fauna changes when pharmaceutical residues are removed, by ozonation, from treated sewage water entering a river.

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1.4 Problem statements 1. How does the biodiversity of aquatic insects change when sewage water is treated with ozonation before entering the river? 2. How does the density of Chironomidae (nonbiting midges), Isopoda, Trichoptera (caddisfly) and Simuliidae (black fly) in the river Knivsta change when sewage water is treated with ozonation before entering the river? 3. Are there any aquatic insects that can only be found in the treated respectively untreated water?

2. Method and materials 2.1 Method

Knivsta sewage treatment plant (STP) was renovated during 2013/2014 to be able to clean

pharmaceuticals from sewage water. In June 2015 this STP was the first full scale STP with

ozonation in Sweden. Studies have shown that pharmaceutical levels will be reduced between

80 and 99.9% (Jerker Fick personal commentary 2016). Benthic organisms included in this

study were obtained from the river Knivsta, Knivsta, located south of the city Uppsala.

Invertebrate samples were collected from six sites (5-10) shown in figure 1 using the kick-

sampling method with five subsamples in each site per sampling event (Appendix 1). Kick-

sampling means that a net is “kicked” along the riverbed for a specific distance (here 50 cm)

and if the ground is to muddy, gently dragged along the ground. The content collected using

the kick-sampling method were put in plastic containers and marked with site number and

date. All of these sites were located downstream from the sewage treatment plant. 30 samples

were collected in spring (April-May) 2015, before the ozonation treatment began, and 30 in

the autumn (October-November) 2015 while the ozonation treatment was in progress.

Figure 1. Map of river Knivsta with sample locations marked. The red square indicates where the sewage

treatment plant is located with samples numbered from 5-10 placed downstream from the STP.

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After the sample collection from the river, the content of each container were analyzed at

Umeå University using a stereo microscope. All invertebrates found in each container were

put in different small glass vials depending on which taxon they belonged to. For example, all

chironomids from the same sample were put in the same vial. A 70% ethanol solution was

added to every glass vial to conserve the invertebrates. Furthermore, all invertebrates were

counted and assigned to categories of each taxa, sample and date.

2.2 Statistical analysis

All statistical analysis were made in Microsoft Excel with a 95% confidence interval for

significance. If the data was normally distributed, T-tests and one way ANOVA’s were used to

investigate changes between species composition before and during the ozonation treatment.

When data were not normally distributed, the non-parametric Kruskal–Wallis one-way

analysis of variance were used. Firstly all data had to be ranked from 1-60. All tied values

with the same number had to be averaged to get the rank they would have received had they

not been tied. N is the total number of observations across all groups. ni is the number of

observations in group i and the ri is the average rank of all observations in group I.

Figure 2. Calculations for the Kruskal-Wallis one-way analysis.

To calculate the diversity index in all the samples the Shannon-Wiener diversity index were

used.

Figure 3. Calculations for the Shannon-Wiener diversity index.

Thereafter a one way ANOVA could be used to calculate the differences in diversity before

and during the ozonation treatment.

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3. Results

3.1 Biodiversity

There was a higher biodiversity at site 10 before ozonation treatment (1.79 ± 0.07) compared

to during the treatment (1.31 ± 0.17) and this difference was significant (F = 21.86, p =

0.002). At site 7 the opposite pattern was found with a higher biodiversity during the

treatment (1.34 ± 0.03) than before (1.12 ± 0.09) and this was marginally significant (F =

5.12, p = 0.054; fig 4). No other significant results could be found when analyzing

biodiversity before and after removal of pharmaceuticals.

Figure 4. Shannon-Wiener diversity index for site 7 and 10 both before and after of removal of pharmaceuticals in

sewage water. Error bars denote ± 1 standard error (SE).

3.2 Invertebrate density

More Gastropoda were found during ozonation treatment (1.47 ± 0.32) compared to before

the treatment (0.2 ± 0.09) when analyzing all sites collectively. This difference was

significant (p = <0.004; Fig 5). Other taxa showed no significant difference when all sites

were compared together.

Figure 5. Mean values of Gastropoda sample both before and during ozonation treatment. Error bars denote ± 1

standard error (SE).

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There were considerable more Gastropoda during ozonation treatment at three different sites

compared to before the treatment. At site 6 no Gastropoda were found before ozonation

treatment. The average number of Gastropoda per sample during ozonation treatment was 3

± 0.632 and this increase was significant (p= 0.003). Similarly, no Gastropoda were found

before ozonation treatment at site 7 either. The average number of Gastropoda per sample

during ozonation treatment was 3.4 ± 0.927 and this difference was also significant (p=

0.003). At site 10 an increase from 0.6 ± 0.4 to 2 ± 0.447 could be found. This difference was

marginally significant (p = 0.06; Fig 6).

Figure 6. Mean values of Gastropoda sample both before and during ozonation treatment. Error bars denote ± 1

standard error (SE).

No significant difference could be found when comparing Isopoda for all sites collectively.

The same trend was found for Isopoda with more Isopoda found at both site 5 and 6 during

ozonation treatment compared to before the treatment. The average number of Isopoda per

sample at site 5 increased from 2 ± 1. 05 to 9.2 ± 1.93 and this difference was significant (F =

10.71, p = 0.011). At site 6 the average number of Isopoda per sample was 1.4 ± 0.24 before

removal of pharmaceuticals and increased to 5.4 ± 1.69 during the ozonation treatment. This

difference was also shown to be significant (F = 5.48, p = 0.047; Fig 7).

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Figure 7. Mean values of Isopoda/sample both before and during ozonation treatment. Error bars denote ± 1

standard error (SE).

No significant differences could be found when looking at Chironomidae, Trichoptera and

Simuliidae per site, nor when comparing all sites collectively. Anisoptera (dragonflies) were

only found before the ozonation treatment but only two individuals in total. Five taxa were

only found in the ozonation treated water ranging from 1-6 individuals: 4 Tipulidea (crane

flies), 3 Corixidae, 6 Lepidoptera (moths and butterflies), 1 Nepidae (water scorpions) and 1

Zygoptera (damselflies). In total 15 taxa were found before ozonation treatment and 19 taxa

during the treatment (Appendix 2).

4. Discussion

4.1 Biodiversity

No clear patterns could be found when studying biodiversity in effluent water from a STP

before and during ozonation treatment. When we compared site to site, a difference could be

found at two different sites, 7 and 9. However, those two results differed in the direction of

the effect as site 7 had a higher biodiversity during the treatment and site 9 before the

treatment. This study suggests that biodiversity isn’t affected in a clear way by

pharmaceutical compounds in aquatic systems. Most likely those relatively low

concentrations (ng-µg/L) of pharmaceutical in the environment are not lethal and thus no

difference in diversity could be found. Toxicology studies often use pharmaceuticals at

concentrations around 1-300mg to test mortality of different organisms when exposed to

pharmaceuticals (Nunes, Carvalho and Guilhermino 2005, Nalecz-Jawecki and Persoone

2005 and Coelho et al. 2011). If some species or groups of species are affected more than

others they might slightly reduce population size but then a niche is available for other

species to occupy which are not as affected by the pollution. This would not show when

analyzing the diversity in an area and therefore more studies on how different taxa’s

behaviors changes when exposed to pharmaceuticals are surely needed.

The sampling of data before and during ozonation treatment wasn’t collected at the same

time of the year. One was collected in spring and the other in autumn, which might cause a

temporally induced difference in the data. Some of the insects may live in the stream as

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larvae and then leave when they are fully developed, if they undergo this change during

summer or early autumn this may cause quite the difference in population size. However,

between November and April, as were sample-times in this study, the production of biomass

and change in biotic community in streams are very low due to low temperatures. Thus no

significant changes due to temporal effects should occur during this time a year. Although if

many species developed into their adult stage during summer and left the river, a lower

number of species may be found in the autumn in comparison to what would have been

found during spring. Ivkovic et al. (2013) states that an increase in water temperature and

length of sunlight is two important factors when studying emergence of aquatic invertebrates.

Many aquatic insects tend to emerge from the river when water temperatures exceeds 16◦C. If

the sampling of data would have been in spring 2 subsequent years a difference in

biodiversity might have been found. Mainly due to a longer ozonation treatment. It is unclear

if 3 months of ozonation is enough to remove all pharmaceuticals from the water and the

sediment. Thus a longer period of ozonation would have been preferred before the sampling

began.

4.2 Invertebrate density

More Gastropoda during ozonation treatment than before could be found when comparing

all sites downstream the STP. The same result was found in 3 of the 6 sites when comparing

changes within sites. Gastropoda is clearly affected by, at least some, pharmaceuticals in their

environment. Fluoxetine, an anti-depressant commonly known as Prozac and Sarafem is only

one of many selective serotonin reuptake inhibitors (SSRIs) that are used widely today.

Serotonin exist in the nervous system among many vertebrates and invertebrates. SSRIs

inhibits the reuptake of serotonin thus increases the serotonin neurotransmission which

affects many physiological functions in mollusks like Gastropoda (De Lange et al. 2006). It

also affects reflexes like heartbeat rhythm and behaviors like feeding, locomotion patterns

and reproduction (De Lange et al. 2006 and Daughton and Ternes 1999). Fluoxetine may

affect behavior at very low concentrations (100ng/L) which is observed in STP effluents (De

Lange et al. 2006). Consequently if low concentrations of pharmaceuticals may have such a

big effect on organisms in the environment the lack of Gastropoda before removal of

pharmaceuticals in this study may be explained by the presence of pharmaceuticals in this

aquatic system. When the ozonation treatment began and the water became less polluted,

organisms that are sensitive to pharmaceutical and other pollutants might have migrated

downstream or upstream to get closer to the cleaner water near the STP.

Freshwater Gastropoda may be used as biological indicators. Some radioactive materials and

heavy metals are concentrated in their flesh and shell (Hart and Fuller 1974). Therefore, it

would be interesting to study if pharmaceuticals also concentrate in their body like other

pollutants. Gastropoda and other mollusks could be interesting subjects to study in the future

and more studies would be necessary to determine if they may be used as indicators for

pharmaceutical and other similar pollutants.

Significantly more Isopoda were found at 2 sites (5 and 6) during ozonation treatment in

comparison to before. De Lange et al. (2009) showed that low concentrations (1-100ng/L) of

ibuprofen, fluoxetine and carbamazepine had an effect on G. pulex. An increase in ventilation

(resting) could be found. G. pulex are related to Isopoda on a class level, both belong to

Malacostraca. Therefore there may be similar effects on Isopoda that might explain this

result. An increase in resting can lead to lower food intake which in turn leads to lower fitness

and less reproduction.

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No significant result could be found when studying the number of individuals for many other

species groups like Chironomidae (nonbiting midges), Trichoptera (caddisflies) and

Simuliidae (black flies). This does not mean that these taxa are unaffected by

pharmaceuticals, but that the number of individuals in this study did not change

significantly. Other biological endpoints such as behavior might have been affected. Studies

have shown that fish behavior can change due to pharmaceuticals in their environment. For

example, European perch (Perca fluviatilis) were shown to become bolder, more active and

less social in the presence of oxazepam (Brodin et al. 2013 and Brodin et al. 2014). This may

also be the case for some invertebrates and thus no changes in quantity could be found. Even

though no direct changes were found for many species groups pharmaceutical may bio

accumulate in invertebrates who gets exposed to those compounds during a long time. Later

if they get eaten by fish for example these compounds will affect fish’s as well. Some

pharmaceuticals which increase feeding rate for consumers might cause an even higher

increase in bioaccumulation with a higher food intake (Brodin et al. 2014). Biomagnification

in aquatic systems can therefore be an important factor to keep in mind when studying

pharmaceuticals in aquatic systems. Due to bioaccumulation the level of pharmaceuticals in

surface water does not directly have to represent the levels found in organisms (Brodin et al.

2014).

More taxa could be found during the ozonation treatment compared to before. Those taxa

occurred only in small quantities ranging from 1-6 individuals per taxa. Therefore it is hard to

know if those taxa are especially sensitive to pharmaceutical compounds or if these

differences are natural random occurrences. Studies with larger sampling sizes are hence

needed to exclude such anomalies.

Lürling, Sargant and Roessink (2005) showed that Daphnia pulex, which is a freshwater

grazer showed an increase in reproduction when exposed to the pharmaceutical

carbamazepine. They were also larger and matured and reproduced earlier. So this species

showed an increase in individuals due to pharmaceuticals. These various responses that

different organisms tend to have as an effect of pharmaceuticals make it hard to predict

general responses of how organisms are affected when exposed to pharmaceuticals.

Ecosystems are diverse and complicated systems which makes it extremely hard to

understand all the processes and interactions therein. Pharmaceuticals have been used

during the last century and especially the last 50 years (Rowland et al. 2012). Therefore,

ecosystems have had been exposed to those compounds for much longer than we have

studied its effects. The removal of pharmaceutical with different removal techniques like

ozonation may have a greater effect on ecosystems than expected. More studies are therefore

needed to assess the effects which the removal of pharmaceuticals has on both different taxa

and on the interactions between species in aquatic systems.

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Pollution 165: 250-258.

Appendix 1

Table 1. Coordinates in DMS format (degrees minutes seconds) for sampling sites 5-10 in river Knivsta.

Sampling site Latitude (DMS) Longitude (DMS)

5 59 43'05.8'' 17 47'28.1''

6 59 43'4.1'' 17 47'29.2''

7 59 43'0.4'' 17 47'32.2''

8 59 42'17.5'' 17 47'31.7''

9 59 41'11.9'' 17 47'02.7''

10 59 39'38.7'' 17 45'06.8''

Appendix 2

Table 2. Number of individuals belonging to each species group and the total number of individuals found both

before and during ozonation treatment in river Knivsta.

Taxa Before During

Chironomidae (Nonbiting midges) 604 995

Isopoda 138 170

Ephemeroptera (Mayfly) 22 11

Trichoptera (Caddisfly) 101 99

Zygoptera (Damselfly) 0 1

Odonata (Dragonfly) 2 0

Simuliidae (Black fly) 42 94

Plecoptera (Stonefly) 19 2

Amphipoda 33 55

Bivalvia (Mussel) 291 162

Coleoptera (Beetle) 2 2

Copepoda 22 4

Acari (Tick & mite) 9 2

Hirudinea (Leech) 92 117

Calopterygidae (Broad-winged damselfly) 5 16

Gastropoda (Snail & slug) 6 44

Tipulidae (Crane fly) 0 4

Corixidae (Water boatmen) 0 3

Lepidoptera (Moth & butterfly) 0 6

Nepidae (Water scorpions) 0 1

Total number of individuals 1388 1788

Dept. of Ecology and Environmental Science (EMG)

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