| Abstract | Zbog sumnje na negativan utjecaj na pčele Europska je unija 2013. privremeno, a potom
2018. godine trajno zabranila uporabu tri djelatne tvari iz skupine neonikotinoida:
imidakloprid, tiametoksam i klotianidin. Zabrana se odnosila na sve načine korištenja, osim
u trajnim staklenicima i za biljke koje cijelu vegetaciju provode u zatvorenom prostoru.
Nedostatak alternativnih, ekotoksikološki povoljnijih insekticida učinkovitih u tretiranju
sjemena ratarskih kultura, doveo je do povećane folijarne primjene insekticida iz starijih
skupina, pa se stoga opasnost za pčele nije smanjila. Upravo se zato istraživanja
provedena u sklopu ove disertacije temelje na dvije hipoteze. Prva je hipoteza da zabrana
neonikotinoida u Hrvatskoj, u područjima u kojima prevladava ratarska proizvodnja, neće
rezultirati pozitivnim učincima na pčelinje zajednice jer su gubitci pčelinjih zajednica u tim
područjima u granicama uobičajenih i prihvatljivih sa stajališta pčelarske proizvodnje (do 10
% godišnje). Druga je hipoteza da su alternativni insekticidi spinosad, klorantraniliprol i
azadiraktin, primijenjeni tretiranjem sjemena kukuruza i šećerne repe, zbog dobrog učinka
na štetnike prihvatljivi kao zamjena za neonikotinoide. Anketnim istraživanjem u Tovarniku,
među ratarima i pčelarima od 2014. do 2016. godine, između ostalih prikupljeni su i podatci
o korištenim insekticidima prije i nakon zabrane neonikotinoida, te o zimskim gubitcima
pčelinjih zajednica i gubitcima tijekom vegetacije. Tijekom 2015. godine u Tovarniku je došlo
do pomora pčela. Podatci o uzrocima pomora dobiveni su od ovlaštenog laboratorija za
određivanje rezidua Hrvatskog veterinarskog instituta u Zagrebu. Temeljem podataka o
količinama primjenjenih insekticida po jedinici površine, izračunati su i indeksi tretiranosti
insekticidima za sve zasijane kulture. Učinkovitost alternativnih insekticida primijenjenih
tretiranjem sjemena kukuruza i šećerne repe na štetnike ratarskih kultura utvrđivana je
laboratorijski. Rezultati anketa obrađeni su standardnim statističkim metodama utvrđivanja
srednje vrijednosti i varijabilnosti (ANOVA) (ARM 9, Gylling Data Management), a
učinkovitost alternativnih insekticida na žičnjake izračunata je po Abbottu (1925) na temelju
mortaliteta kukaca. Podaci o učinkovitosti obrađeni su statistički faktorijelnom analizom uz
pomoć ANOVE i rangirani testom rangova po Duncanu (ARM 9, Gylling Data Management).
S obzirom da je mortalitet pipa i repina buhača u pokusima očitavan u vremenskim
intervalima od 24 sata, podaci o broju živih jedinki izloženih djelovanju korištenih insekticida
obrađeni su primjenom metoda analize preživljavanja Kaplan Majerovom metodom u R
programskom okruženju, korištenjem specijaliziranih paketa za deskriptivnu i inferencijalnu
statističku analizu (paketi „survival“, „ggsurvfit“ i „survminer“) . Podatci dobiveni anketama
pokazuju da je na području Tovarnika dominantna ratarska proizvodnja (na 97,32 %
površina) te da je samo sjeme šećerne repe kroz sve godine provedbe ankete tretirano
neonikotinoidima (privremena se zabrana nije odnosila na ovu kulturu), dok je sjeme
suncokreta neonikotinoidima tretirano u vegetacijskoj sezoni 2012/2013. godine, prije
njihove zabrane. Također, podatci pokazuju značajno veće prinose kod suncokreta
posijanog iz tretiranog sjemena (4,20 t/ha) u odnosu na suncokret posijan iz netretiranog
sjemena (2,56 t/ha). Pojavom visokih populacija štetnika povećani su i indeksi tretiranosti
insekticidima kod šećerne repe (2,9) u 2012/2013. i 2013/2014., strnih žitarica (0,97) u
2012/2013. i (0,99) u 2014/2015. te uljane repice (2) u 2014/2015. Ekspertne procjene
indeksa tretiranosti za ove kulture su 2,5; 0,2; 1. Zimski su gubitci pčelinjih zajednica bili u
skladu s očekivanim i prihvatljivim gubitcima (6,02 % u 2013/2014., 8,79 % u 2014/2015. i
11,54 % u 2015/2016). Do pomora 33 % pčelinjih zajednica u Tovarniku tijekom paše u
2015. godini došlo je zbog tretiranja suncokreta pripravkom na osnovu djelatnih tvari
klorpirifos i cipermetrin, koje nisu imale dozvolu za primjenu na ovoj kulturi. Faktorijelna
analiza pokazala je da učinkovitost ovisi o vrsti insekticida, dozi i o vremenu tijekom kojeg
su kukci izloženi insekticidima. Od alternativnih insekticida primijenjenih tretiranjem
sjemena, klorantraniliprol, spinosad i azadiraktin su pokazali zadovoljavajuću učinkovitost
na repina buhača. Zadovoljavajuća je i učinkovitost spinosada na žičnjake u kukuruzu. |
| Abstract (english) | Neonicotinoids have been used since the 1990s to protect against pests and are now the
most widely used insecticides in the world. This group of insecticides includes seven active
ingredients: imidacloprid, thiamethoxam, clothianidin, thiacloprid, acetamiprid, nitenpyram
and dinotefuran. They are applied foliar over the leaves, by seed treatment and application
in the form of granules. About 60% of the neonicotinoids produced are used specifically for
seed treatment and in the form of granules. Although the insecticides in this group have low
acute toxicity to mammals, birds, and fish, negative phenomena are associated with the use
of neonicotinoids on bees. Bees exposed to sublethal doses of this insecticide may develop
various problems (e.g., flight and orientation problems, loss of sense of taste and foraging
ability), and their ability to overwinter is reduced. The active ingredients imidacloprid,
clothianidin, dinotefuran and thiamethoxam are very toxic to bees, while the active
ingredients thiacloprid and acetamiprid are moderately toxic to bees. The problem of the
decline or loss of bee colonies is present worldwide. For example, between 1981 and 2005,
the number of bee colonies in the United States declined from 4.2 to 2.4 million, and in
southern Europe there was a loss of about 40% of bee colonies. Colony Collapse Disorder
(CCD) is also associated with the use of insecticides from the group of neonicotinoids, such
as the negative effects of harmful organisms on bees Varroa destructor Oudemans, the
appearance of fungal diseases and viruses, the reduction of pasture area and problems
with the queen. In 2013, the European Commission temporarily banned the use of three
insecticidal active ingredients from the neonicotinoid group (imidacloprid, thiamethoxam
and clothianidin) for two years due to suspicions that insecticides from this group have
harmful effects on bees. The ban covered the treatment of seeds and soils for crops that
attract bees and spring cereals, while the authorization remained valid for the treatment of
winter cereals and sugar beet seeds and for use in protected areas, as well as for foliar
treatment after flowering. The final ban was issued by the European Commission on April
27, 2018, on the recommendation of the European Food Safety Agency (EFSA) based on
a thorough review of all relevant research. The decision refers to a complete ban on the use
of imidacloprid, thiamethoxam and clothianidin, except in permanent greenhouses, and the
plants obtained in this way remain in a permanent greenhouse throughout their lifetime. The
ban on the use of three insecticidal active ingredients from the neonicotinoid group has
raised concerns about the potential impact on agricultural production.
Surveys conducted immediately after the temporary ban showed that in some crops, such
as oilseed rape, pest infestations increased significantly, more insecticides (mainly from the
pyrethroid group) were applied foliar, and yields decreased significantly (by up to 15%).
The main aim of this doctoral thesis was to determine the impact of the ban on the use of
active substances from the group of neonicotinoids, widely used as insecticides in seed
treatment, on the occurrence of pests in agricultural production and on the condition of bee
colonies in eastern Slavonia, a part of Croatia with intensive arable production. Because
there are few insecticidal active ingredients that can be used for seed treatment of field
crops, it was necessary to investigate the efficacy of alternative, more ecotoxicologically
favourable insecticides that could be considered as substitutes for the banned
neonicotinoids. Precisely for this reason, the research conducted in this dissertation is
based on the main hypothesis that the ban of neonicotinoids in Croatia does not have a
positive impact on bee colonies in areas where arable production is predominant, because
the losses of bee colonies in these areas are within normal and acceptable limits from the
point of view of bee production (up to 10% per year). The second hypothesis was that the
alternative insecticides spinosad, chlorantraniliprole and azadirachtin, used in seed
treatment of corn and sugar beet, are acceptable substitutes for neonicotinoids due to their
good effect on pests. To prove the established hypotheses, the following objectives were
set for this study: (a) Analysis of data on crop area, pest incidence and damage, crop
protection measures, insecticide treatment index, and type of insecticides used before and
after the ban on neonicotinoids; (b) Analysis of data on the number of bee colonies, type of
bee pasture and supplemental feeding, treatment of bee colonies for diseases and pests,
number of bee colonies in the fall and number of bee colonies after wintering in the spring
during the period studied, and losses and causes of losses of bee colonies during wintering
and vegetation, (c) Determination of the type and level of insecticide residues in dead bees
in pest infestations associated with insecticide use, (d) Determination of the efficacy of
spinosad, chlorantraniliprole, and azadirachtin in seed treatments against important pests
of arable crops: corn and sugar beet.
In Croatia, arable crops and gardens account for more than half of the total arable land, and
the main agricultural crops considered in this study are wheat, barley, corn, sugar beets,
oilseed rape, sunflower, and soybeans. Since pests have the highest damage potential
(18%) next to weeds, the dissertation also deals with the most important pests of arable
crops, paying special attention to the wireworms, the sugar beet weevils and the sugar beet
flea beetles as pests that were used in laboratory trials during the research. In addition to
agricultural production, beekeeping is also of great importance. In addition to the direct
benefits that bees bring through the production of honey and other bee products, they also
create indirect benefits through the pollination of plants (they pollinate about 80% of
flowering plants and, together with other pollinators, more than 70% of the plants that feed
humans). In addition to the negative effects of pesticides, bees are susceptible to various
diseases and pests, and poor beekeeping practices, queen problems, and a lack of highquality
forage and less pasture (nectar and pollen) can also negatively impact bees. Losses
of bee colonies can occur in winter or during the season, i.e. during grazing. In 2008/2009,
winter losses in Croatia amounted to 13.16%, in 2012/2013 to 9.5% and in 2015/2016 to
16.4%. After the ban on neonicotinoids, the number of insecticides for seed treatment and
foliar application declined, so that today there are no approved insecticides for treating
sunflower seed to protect against wireworm, for treating corn seed to protect against
western corn rootworm, and for treating sunflower and sugar beet seed to control aphids.
Alternative insecticides, i.e., candidates that could replace the banned neonicotinoids, are
chlorantraniliprole, spinosad, and azadirachtin. Chlorantraniliprole is a systemic insecticide
with low bioconcentration potential and low toxicity that is selective for beneficial arthropods,
making it suitable for use in integrated pest management. Spinosad is a fermentation
product from the soil bacterium Saccharopolyspora spinosa Mertz and Yao. It belongs to
the group of biological insecticides, naturalites, and is less toxic to beneficial insects than
other insecticides. It has low toxicity to mammals and the environment. Azadirachtin is a
biological insecticide obtained as a product from the neem plant (Azadirachta indica Juss).
It has a negative effect on the feeding, development, and reproduction of pests, and when
used properly, it is not expected to have harmful effects on humans, wildlife and the
environment, as well as on other beneficial insects.
The dissertation included surveys of farmers and beekeepers, as well as laboratory testing
of the efficacy of alternative insecticides and analysis of data on the causes of bee mortality
obtained from an approved laboratory for residue determination. In Tovarnik, a town in
eastern Croatia, a survey of farmers and beekeepers was conducted from 2014 to 2016.
Since bee losses occurred in Tovarnik during the 2015 growing season, data on the causes
of the losses were analysed, which came from the approved laboratory for residue
determination, the Croatian Veterinary Institute in Zagreb. Laboratory investigations were
carried out in 2016 and 2017 to assess the effectiveness of spinosad, chlorantraniliprole,
and azadirachtin as treatments for corn seeds against wireworms. Additionally, in 2017, the
same compounds were examined for their efficacy in treating sugar beet seeds against the
sugar beet weevil and sugar beet flea beetle. The results of the surveys were processed
using standard statistical methods to determine mean and variability (ANOVA) (ARM 9,
Gylling Data Management), and the efficacy of the alternative insecticides was calculated
on the basis of wireworm mortality according to Abbott (1925). The efficacy data were
processed using statistical factor analysis (ANOVA) and ranked using the Duncan rank test
(ARM 9, Gylling Data Management). Since the mortality of sugar beet weevil and sugar
beet flea beetle in the experiments was read in 24-hour time intervals, the data on the
number of living individuals exposed to the effects of the insecticides used were processed
with the Kaplan-Mayer method of survival analysis in the R programming environment using
special packages for descriptive and inferential statistical analysis (packages "survival",
"ggsurvfit" and "survminer").The results of the surveys among farmers show that agricultural
production dominates in the Tovarnik region (on 97.32% of the total agricultural area), and
the questionnaire covered most of this area (71.87% in 2012/2013, 72.38% in 2013/2014
and 90.36% in 2014/2015). In all survey years, only sugar beet seed was treated with
insecticides from the neonicotinoid group on all croplands (the temporary ban did not apply
to this crop), while sunflower seed was treated with the same insecticides in the 2012/2013
cropping season (on 73.99% of the croplands of this crop), before the ban on the use of
insecticides from this group came into effect. Of the other active substances, during the ban
on the use of neonicotinoids, corn seeds was treated with an insecticide from the pyrethroid
group (e.g. tefluthrin) and sugar beet seed was treated with a combination of neonicotinoids
(the ban on use did not apply to this crop) and pyrethroids.Appropriate pneumatic seeder
were used for sowing neonicotinoid-treated seed in both crops (an exception was sugar
beet seed in a 4-ha plot in 2014, where a mechanical seeder was used). In certain growing
seasons, pests of small grains (cereal leaf beetle), sugar beets (sugar beet weevil),
soybeans (painted lady) and oilseed rape (cabbage stem weevil, common pollen beetle)
were controlled on a leaf-by-leaf basis, with preparations containing active ingredients from
the neonicotinoid group not being used. Due to the occurrence of high pest populations,
insecticide treatment indices increased in the 2012/2013 and 2014/2015 growing season in
sugar beets (2,9), small cereals (0,97) in 2012/2013 and (0,99) in 2014/2015, and canola
(2) in 2014/2015. Expert estimates of the treatment index for these crops are 2.5; 0.2; 1.
Yield data for sunflower sown from neonicotinoid-treated seed only showed significantly
higher yields (4.20 t/ha) compared to sunflower sown from untreated seed (2.56 t/ha).
The survey of beekeepers included most colonies in Tovarnik (83.52% in 2013, 90.32% in
2014 and 86.67% in 2015). The bees collected nectar and pollen according to the pasture
plan in the area, and the beekeepers fed with sugar syrup and sugar cakes. Winter losses
of bee colonies were in accordance with expected and acceptable losses of bee colonies
(6.02% in 2013/2014, 8.79% in 2014/2015 and 11.54% in 2015/2016). In the 2013 season,
the loss of three bee colonies was reported, and in 2015, the death of 33 % bee colonies
was reported. The results of the approved laboratory show that the cause of the loss of bee
colonies during grazing in 2015 was the treatment of sunflowers with the product in a
combination of the active ingredients chlorpyrifos from the group of organophosphorus
insecticides and cypermethrin from the group of pyrethroids, although this product
(Chromorel-D) had no approval for use on sunflowers.
Laboratory studies show that chlorantraniliprole (efficacy less than 15%) and azadirachtin
showed no efficacy against wireworms in the treatment of corn seed, while spinosad is very
effective at dosages of 3.5 and 5 mg/kg seed (efficacy about 70%). Chlorantraniliprole,
spinosad, and azadirachtin showed low efficacy against sugar beet weevil (in terms of pest
mortality). Chlorantraniliprole showed good efficacy against the sugar beet flea beetle in
treating sugar beet seed (pest mortality at a dose of 0.2 mg and at a dose of 0.6 mg).
Spinosad also showed excellent results against this pest at all doses. In addition,
azadirachtin also showed excellent efficacy against sugar beet flea beetle at all doses.
Factorial analysis of laboratory test results has shown that insecticide efficacy depends on
the type of insecticide, dose, and duration of insecticide exposure. |
