Fish

Question 1

Microplastic particles can be transported from

Answer 1

freshwater to marine habitats
and have been detected in more than 100 fish species around the
world

Question 2

Laboratory studies have revealed

Answer 2

the negative
impact of microplastics in fish; such as intestinal damage and
inflammation from short to long term exposure

Question 3

microplastics can enter

Answer 3

internal organs after adhering to the gills
and skin

Question 4

There is widespread recognition that wetlands provide

Answer 4

valuable ecological services including habitat for species, protection against
floods, water purification, amenities and recreational opportunities

Question 5

The sex of adult in-
dividuals can be easily distinguished on the basis of externally

visible characteristics (Leusch et al., 2006), which allows for
gender-dependent investigations in pollution monitoring.

Answer 5

on the basis of externally visible characteristics (Leusch et al., 2006), which allows for
gender-dependent investigations in pollution monitoring

Question 6

some urban wetlands are specifically
designed to treat

Answer 6

urban runoff and to receive stormwater which
makes them more vulnerable to environmental stress via anthro-
pogenic influence and contamination

Question 7

we know that microplastics can
occur

Answer 7

in natural wetland sediments, waters and birds However, monitoring of plastic levels in urban wet-
lands and their inherent taxa is uncommon. Urban wetlands receive
a considerable amount of material carried by surface run-off, and
therefore constitute an important source of microplastic pollution

Question 8

In addition, urban wetlands are typically

Answer 8

small water bodies that are heavily influenced by surrounding
catchment land use. The seriousness of microplastic pollution in
small water bodies has been highlighted in some regional case studies

Question 9

Gambusia holbrooki (Eastern mosquitofish) is invasive in

Answer 9

mostcontinents under a wide range of temperate climate conditions.
Because of its widespread distribution, high abundance, ease of
capture and ability to adapt to laboratory settings, Gambusia hol-
brooki is a common model fish for ecotoxicological testing and
environmental monitoring

Question 10

The accumulation of metals and persistent organic pollutants has been observed in Gambusia holbrooki

Answer 10

while few studies have investgated microplastic accumulation

Question 11

Facts about Gambusia holbrooki

Answer 11

• an invasive species in Austrlia
• has ahigh level of biomass and dominance in some wetlands and small
  water bodies
• are a hardy species and are often found inhabiting polluted artificial
  ponds and wetlands where native species do not occur
• biomonitoring and routine investigations
  based on Gambusia holbrooki has limited impact on understanding
  pollutant effects on local, native fish populations, but does provide
  valuable information regarding impacts on resident species within
  polluted ponds and wetlands.
• Given that microplastic research is substantially lacking for
  freshwater biota in the Southern Hemisphere, we conducted an
  investigation to determine the baseline pollution level of micro-
  plastics in Gambusia holbrooki. Fish size, weight and gender

Question 12

The greater Melbourne metropolitan area supports a population
of

Answer 12

approximately 4.5 million and encompasses a catchment area of
approximately 12,800 km2 ,

It contains a complex network of rivers, streams and constructed wetland sys-
tems.

These wetlands are constructed for the interception and
treatment of stormwater, to attenuate flows during storms and to
treat water quality.

Question 13

Recording the colour was not considered necessary because

Answer 13

The digestion process can discolour the particles. Based on their
morphology, microplastics were classified into four shapes: fiber,
film, fragment or pellet

Question 14

The specific polymer composition was identified under
All spectra were compared
with a database from Bruker for verification. Spectra matching with
a quality index more than 70% were accepted and the abundance of

microplastics was re-calculated by removing all the verified non-
plastics. Of all 194 visually identified items, 109 items were veri-
fied. Such a validation rate (57.6%) was higher than the generally

accepted level (50%)

Answer 14

the attenuated total reflec-
tion (ATR) mode of a micro-Fourier Transform Infrared
Spectroscopy (m-FT-IR, Bruker, LUMOS)

Question 15

All spectra were compared

Answer 15

• with a database from Bruker for verification.
• Spectra matching with
  a quality index more than 70% were accepted and the abundance of microplastics was re-calculated by removing all the verified non-plastics.
• Of all 194 visually identified items, 109 items were verified. Such a validation rate (57.6%) was higher than the generally accepted level (50%)

Question 16

To evaluate any background contamination we set up

Answer 16

one blank control for every three samples, totaling 120 blanks.

Question 17

For each blank fish tissues were replaced

Answer 17

• by 2 ml filtered water and analyzed
  simultaneously with other samples.
• Eight items were confirmed in
  blanks which equated to 0.067 items/ind., 0.033 items/g and a
  detection rate of 6.67%.
• We have corrected all our results by sub-
  tracting the mean level of background contamination from those
  blanks to account for this error.

Question 18

Polymer identification in fish body and head:
Of the 109 selected items from fish bodies and heads

Answer 18

• 68 items were confirmed as plastic (62.4%).
• Eleven polymer types were identified: the most common polymers were polyester (25.7%),
  rayon (10.1%),
  polyamide (7.3%) and polypropylene (5.5%) (Sup-
  plementary tables Table S4).
• In particular, polyester and rayon
  occurred in more than 70% of sampling sites.
• Sixty plastic items (72.3%) belonging to 11 polymer types were
  identified from body samples
• 8 plastic items (30.8%) belonging
  to 5 polymer types were identified from head samples (Fig. 2A andB).
• Polyester and rayon were frequently detected in fish bodies
  across all sampling sites while polyester and polyamide were more commonly detected in fish heads
• (mean = 7.2%) of fish showed microplastics in the head (Table 1).

Question 19

The average abundance of microplastics in bodies ranged

Answer 19

• from 0.18 items/ind. to 1.13 items/ind. (0.52 items/g to 4.4 items/g)
• with a mean level of 0.60 items/ind. (1.94 items/g).
• In heads across sites,the average detection rate ranged from 0 items/ind. to 0.28 items/ind. (0 items/g to 12.5 items/g) with a mean of 0.11 items/ind. (3.1
  items/g) (Table 1).

Question 20

The abundance of microplastics
in bodies was

Answer 20

significantly higher than those in heads in terms of
items/ind. (p < 0.05).

Question 21

Fiber was the most common morphology observed in both the

Answer 21

bodies and heads of the fish, comprising 62.5%e100% of micro-
plastics found at each wetland except those from S5 where no
microplastics were identified (p < 0.05) (Fig.

Question 22

Characteristics of microplastics in different parts of fish:
The microplastic sizes ranged from

Answer 22

• 0.09 mm to 4.86 mm in bodies an
• 0.22e2.01 mm in heads.
• Microplastics of a size less than 1 mm were
  prevalent, accounting for 52.9% of microplastics found in fish
  bodies and 83.9% in fish heads (Supplementary materials Fig. S1).
• The microplastics found in the bodies were significantly larger than those found in the heads
• head parts were significantly greater than fish without
  microplastics in the body (p < 0.01)

Question 23

Female individuals contained more

Answer 23

• microplastics in their bodies
  than males (p < 0.05) (Fig. 5 A)
• but not in their heads (p > 0.05)
  (Fig. 5 B).
• Regarding microplastic shapes, female fish contained
  more non-fibers in their bodies than males.

Question 24

microplastic pollution levels in an Australian freshwater fish from the Greater Melbourne Area.

Answer 24

• investigations into the microplastic pollution in
  freshwater organisms are few compared to similar studies of ma-
  rine organisms.
• in addition, this is the first known field study to
  specifically target microplastics in Gambusia holbrooki.
• Based on the available literature, the average abundance of microplastics in freshwater fish, amphibians and invertebrates can range from 0.5 items/ind. to 19.2 items/ind. (Table 2).
• More than 70% of those studies had microplastic detection rates which exceeded 20%.
• In comparison, of the 19.4% of fish found with microplastics in this
  study, there was an average abundance less than 1 items/ind. * *Our results indicate a relatively low prevalence of microplastic pollution in Gambusia holbrooki from the Greater Melbourne Area compared to similar studies completed elsewhere on other species

Question 25

high microplastic detection rates and abundances in fish bodies (p < 0.05) were encountered at

Answer 25

all sites except for S8 located from the North-West of Melbourne’s
central business district.

Question 26

Fish from S1, which was nearest to central
Melbourne

Answer 26

displayed the highest detection rate and abundances of
microplastics in their bodies

Question 27

The North-Western region of the Greater Melbourne Area comprises

Answer 27

traditional industrial zones
wherein the largest stormwater wetlands are located

Question 28

pollution pattern observed in this study indicates

Answer 28

that key environmental pressures from anthropogenic sources may be having an influence on microplastic spatial distribution

Question 29

Because Australian cities have separate storm water and sewage systems,

Answer 29

sewage wastewater discharges should not be a significant
contributor of microplastics to river networks and wetlands.

Question 30

Microplastics recovered from the body and head of Gambusia
holbrooki represent two critical pathways for microplastic uptake;

Answer 30

• one via the gill and the other via the gastrointestinal tract.
• In addition to ingestion, laboratory studies have confirmed that
  microplastics can be passively captured by fish gills (Jabeen et al.,2018).
• Microplastics have also been detected in the gills of marine
  fishes
• In this study, the microplastic abundance, polymer type,
  shape and size significantly varied between the heads and bodies of Gambusia holbrooki.

Question 31

In natural conditions, they can be quickly
removed by

Answer 31

• the filtering process
• while those particles in the
  gastrointestinal tract can persist there for a long time during the
  digestion process (Bath and Eddy, 1980; Maina, 2002). Total sus-
  pended solids (TSS) and turbidity in water may also affect binding
  to gills.

Question 32

Almost all of the microplastics observed in the fish heads were

Answer 32

• fiber and corresponding polymer types
  textiles.
• High abundances of these polymers are commonly re-
  ported in other studies too (Table 2).
• Morphologically, fibers of a flexible nature may more readily entangle with gill filaments. We
  observed high proportions of natural cellulose fibers in fish heads,
  which means that natural fibers such as plant residues can also be captured by gills.
• fibers are more easily entangled with gill struc-
  tures.

Question 33

the dominance of fibers in inland freshwater bodies is

Answer 33

very common, including small water bodies which are the natural
habitats of Gambusia holbrooki

Question 34

Female individuals showed more
ingested microplastics than males. The female individuals caught
were

Answer 34

• generally larger in both size and weight than their male
  counterparts (Supplementary materials Fig. S3).
• Larger body sizes may increase the possibility of ingesting more microplastics as we
  have discussed.
• Secondly, the different feeding behaviors between
  male and female individuals could provide another explanation.

Question 35

Whilst some microplastics are
buoyant, others may sink down because of

Answer 35

• biological and physical
  processes like biofilm formation and degradation

Question 36

One possible explanation for the
greater abundance and diversity of microplastics found in female
fish is that

Answer 36

they may be exposed to greater volumes and different
types of microplastics, whereas male individuals almost exclu-
sively contained fibers.

Question 37

Presuming that the body is
representative of uptake via the gut and the head is representative
of uptake via the gills, we conclude that

Answer 37

• the gut is the predominant
  route of microplastic uptake in this species.
• The overall prevalence
  of microplastics in these fish was 19.4% (body) and 7.2% (head)

Question 38

microplastic uptake depends on ( pg 72/ section 4.3)

Answer 38

• size, weight and gender of Gambusia holbrooki.
• small plastics (e.g. nano-plastics)
• microplastic ingestion was directly proportional to fish size and weight.

Question 39

all of the sampling sites are within the influences from
(Pg 71/ should come after flash card 29.)

Answer 39

• urban intensive land-use.
  *Wetlands,in particular constructed stormwater wetlands, receive a widerange of pollution in storm events (Allinson et al., 2015; Dobbie andGreen, 2013),
• whilst recreational activities in urban wetlands also
  increase the risk of microplastic pollution via unmanaged waste
  disposal and recreational fishing.

Question 40

Fish were rinsed (pg 67//should come after flash card 12)

Answer 40

• with filtered tap water to remove Fish were
  rinsed with filtered tap water to remove any microplastics attached
  to the skin,
• then the heads were removed with a scalpel and the
  head and body sections were weighed separately and stored in
  separate 100 ml glass jars.
• Alkaline hydrolysis was used to denature proteins and hydrolyse
  compounds in the current study

Question 41

Visual assessments
were used to identify microplastics according to (pg 67//should come after flash card 12)

Answer 41

• the observable
  physical characteristics of the particles (Yang et al., 2015). We
  recorded size, shape and the number of microplastic particles