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Environmental Pollution 236 (2018) 416 e 424 Contents lists available at ScienceDirect Environmental Pollution

Contents lists available at ScienceDirect

Environmental Pollution

journal homepage: www.elsevier.com/locate/envpol

journal homepage: www.elsevier.com/locate/envpol What is the aquatic toxicity of saponin-rich plant extracts

What is the aquatic toxicity of saponin-rich plant extracts used as biopesticides? *

Xiaogang Jiang * , Hans Chr Bruun Hansen, Bjarne W. Strobel, Nina Cedergreen

, Hans Chr Bruun Hansen, Bjarne W. Strobel, Nina Cedergreen Department of Plant and Environmental Sciences,

Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

article info

Article history:

Received 7 May 2017 Received in revised form 10 January 2018 Accepted 17 January 2018

Keywords:

Saponins Biopesticides Species sensitivity distribution Aquatic toxicity

abstract

Saponin-rich extracts from Quillaja saponaria and Chenopodium quinoa have been registered by US EPA as active ingredients in biopesticides, and extract from tea seed powder, Camellia oleifera has been proposed for biocidal use. If saponin-rich biopesticides are ef cient against pests, they are most likely also bioactive in the aquatic environment against non-target organisms. The aim of this study was to conduct an effect assessment of saponin-rich plant extracts by using species sensitivity distributions based on acute toxicity tests. The maximal concentrations protecting 95% of the aquatic species (HC 5 ) of saponins extracted from quillaja bark, tea seed coat and quinoa seed coat were 2.91 ± 1.00, 0.22 ± 0.11 and 22.9 ± 5.84 mg/L, respectively. The 100-fold difference in toxicity between the saponin-rich extracts from different plant species, indicate that saponin toxicity depends on the species it origins from, making read-across between saponins a dubious exercise. In addition, the predicted environmental concen- trations of different saponins are close to or higher than their water quality standard, which means that the extracts might pose a risk to the aquatic environment if not used cautiously. © 2018 Elsevier Ltd. All rights reserved.

1. Introduction

Saponins are a class of natural compounds, found abundantly in more than 100 families of plants ( Güçlü-Üstünda g and Mazza, 2007). They are characterized by their structure containing a tri- terpene or steroid aglycone bound to a sugar moiety ( Fig. 1 ). Sa- ponins have antiviral ( Güçlü-Üstünda g and Mazza, 2007 ), antiprotozoal and antibacterial activities ( Potter et al., 2010 ) and are regarded as wormicides ( Szakiel et al., 2005 ), fungicides ( Rubio- Moraga et al., 2011 ) and piscicides ( Cannon et al., 2004 ). These uses indicate that saponins are bioactive and may be used as bio- pesticide ingredients. The de nition of biopesticides by the U.S. Environmental Protection Agency (EPA) is: Biopesticides include naturally occurring substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesti- cides), and pesticidal substances produced by plants containing added genetic material (plant-incorporated protectants). ( U.S. Environmental Protection Agency, 2017 ). Saponin-rich plant

* This paper has been recommended for acceptance by Dr. Harmon Sarah Michele. * Corresponding author. E-mail addresses: jiang@plen.ku.dk (X. Jiang), haha@plen.ku.dk (H.C.B. Hansen), bjwe@plen.ku.dk (B.W. Strobel), ncf@plen.ku.dk (N. Cedergreen).

0269-7491/ © 2018 Elsevier Ltd. All rights reserved.

extracts belong to the rst group, and saponins from Quillaja sap- onaria and Chenopodium quinoa have already registered by U. S. EPA as biopesticides ( U.S. Environmental Protection Agency, 2007,

2005 ).

Saponins from Q. saponaria can be used for control/suppression of pathogenic fungi or plant parasitic nematodes in vegetable or fruit elds using a dose of 1.4 e 3.7 g liquid extract/m 2 (~8.6% of saponins) ( U.S. Environmental Protection Agency, 2007 ). Saponins from tea seed powder or pellets ( Camellia oleifera ) have been used as soil additives with documented growth enhancing effects with 1.5 g powder/m 2 for pot-grown beet, oat, and barley plants ( Andresen and Cedergreen, 2010 ), or have been used to expel earthworms from golf courses and sport elds applying 30 g pel- lets/m 2 once a month ( Potter et al., 2010 ). Saponin extracts from C. quinoa can be used for control of pathogenic fungi on tubers, legume, and cereal seeds, with 1 g liquid/m 2 ( U.S. Environmental Protection Agency, 2005 ). They also showed potential use against snails, with an application dose of 0.6 g coat/m 2 (10 mg saponin/L in the water of rice elds) ( Joshi et al., 2008 ). The saponin-rich ma- terials are often biowaste products. The tea seed pellets are, for example produced during oil production ( Andresen and Cedergreen, 2010 ), while quinoa coat is generated during clean- ing of the quinoa seed for consumption ( San Martín et al., 2008 ). There is a great interest in creating a rational reuse of the biowastes,

X. Jiang et al. / Environmental Pollution 236 (2018) 416e 424

417

et al. / Environmental Pollution 236 (2018) 416 e 424 417 Fig. 1. Structure of quillaja

Fig. 1. Structure of quillaja saponin. R may be either -H or substituted by one or more sugar moieties to form different saponins. The lipophilic part (shown in red) located in the middle of the structure is the aglycone. Modi ed after Fern andez-Tejada et al. (2014) . (For interpretation of the references to colour in this gure legend, the reader is referred to the Web version of this article.)

and the saponin-rich bioproducts seem to have a considerable potential. The saponins in the three plant species contain a tri- terpene aglycone, which means that all saponins have a similar aglycone with different functional groups and sugar chains attached. It is, however, unknown to what extent the naturally produced but different saponins have a similar biological activity. Also, information on their speci c toxicity to a wide range of spe- cies is lacking. Information on toxic concentration ranges and source-speci c saponin toxicity are important, if we are going to use saponin-rich plant extracts in large quantities as biopesticides in an environmentally sustainable way. The very low octanol/water partition coef cient ( k ow ) of sapo- nins and hence poor bonding to organic matter make saponins prone to leaching from soils ( U.S. Environmental Protection Agency, 2007 ). Hence, to know their toxicity to aquatic organisms is important. In Europe, there are two regulatory frameworks giving guidance on how to produce water quality standards. One is Environmental Quality Standards (EQS) for chemicals which are used for chemicals in general and used retrospectively described in the Water Framework Directive (WFD) ( European Commission, 2011 ). Another one is Regulatory Acceptable Concentrations (RAC), which is used prospectively for Plant Protection Products and their Residues ( EFSA PPR, 2013 ). Within both frameworks, several options are possible to derive water quality standard for saponins depending on the data available. One of the more comprehensive and preferred methods is to use Species Sensitivity Distributions (SSDs). SSDs describe the variation among a set of species in the sensitivity towards a certain compound (or a mixture of compounds) by applying a statistical distribution to the data ( Posthuma et al., 2002 ). Based on the distribution, a concentration protecting a certain percentage of the species in the community can be determined. Hence, if we want to protect at least 95% of the species, the environmental concentration must be below the con- centration that is not hazardous to 5% of the species (HC 5 ). The SSD based water quality standards underlying the WFD and Plant Pro- tection Products Regulation both use the median HC 5 derived from the SSD curve and an additional assessment factor (AF) for extrapolation to the eld, but the methodology used is slightly different as well as the terminology. The WFD makes a distinction in AA-EQS (based on chronic toxicity data) and an MAC-EQS (based on acute toxicity data) and for both AA-EQS and MAC-EQS deriva- tion based on SSDs at least 10 valid toxicity data for at least 8 different taxonomic groups are required. For MAC-EQS derivation the median HC 5 from the SSD constructed with acute toxicity data (50% effect or lethal concentrations, LC 50 and/or EC 50 values) and an AF of 10 is selected ( European Commission, 2011 ). In the aquatic

effect assessment underlying the PPP Regulation, a distinction is made between an acute (based on acute LC 50 /EC 50 data) and a chronic (based on chronic No Observable Effect Concentrations NOEC or EC 10 values) Regulatory Acceptable Concentration (RAC s- w;ac and RAC sw;ch , respectively). In deriving the RAC sw;ac and RAC sw;ch by means of the SSD approach at least 8 valid toxicity data for the potentially sensitive taxonomic groups should be provided ( European Commission, 2011 ). For saponins with a more or less biocidal mode-of-action toxicity data of aquatic vertebrates, in- vertebrates, plants and microorganisms may be used to construct the SSD if the toxicity data comprise at least 6 different taxonomical orders/families ( EFSA PPR, 2013 ). Note that in pesticide risk assessment the functions of microorganisms need to be protected and not necessarily their structural properties. For RAC sw;ac deri- vation the median HC 5 from an SSD constructed with acute toxicity data (LC 50 and/or EC 50 values) and an AF of 3 e 6 is selected ( EFSA PPR, 2013 ). The size of the selected AF depends on several criteria, amongst others, on the position of the toxicity data in the tail of the SSD, the goodness-of- t of the SSD curve, the steepness of the SSD curve, the toxicity data used and the 95% con dence limit around the HC 5 estimate. This study will use both the SSD derives MAC-EQS and RAC sw;ac estimates to assess the potential risks of saponin-rich plant extracts to the aquatic environment. The aim of this study was, therefore, threefold: rst, to test the sensitivity of non-target aquatic species to saponin-rich plant ex- tracts to nd the more sensitive species among plants, algae, worms, mollusks, arthropods, sh embryo and bacteria. Secondly, to test whether the bioactivity of the saponin-rich extracts of different botanical origin based on triterpene sapogenins was similar, making read across between saponins possible and thirdly, to determine the MAC-EQS and RAC sw,ac for the three different saponin-rich plant extracts and to evaluate them in relation to the Predicted Environmental Concentration (PEC).

2. Materials and methods

2.1. The tested saponin-rich plant extracts

Three kinds of saponin crude extracts from quillaja bark, tea seed coat, and quinoa seed coat were tested in this study, together with one pure quillaja saponin mixture, which was included as a reference and used as a standard for tentative quanti cation of saponin content of the extracts. The sources of the three saponin crude extracts were: quillaja extract: S7900 ® from Q. saponaria bark (Sigma-Aldrich), tea extract: water extract of tea seed coat powder from C. oleifera (NOR-ADD A/S, Denmark), quinoa extract: water

418

X. Jiang et al. / Environmental Pollution 236 (2018) 416e 424

extract of quinoa seed coat powder, a Real -type variety originating from Bolivia ( Ruiz et al., 2017 ). Water extraction was conducted by adding 5 g tea or quinoa seed coat powder to 50 mL MilliQ water. After 20 min stirring (using a 10 mm magnetic stirrer at 100 rpm/min), the supernatant was passed through a lter paper (Whatman ® quantitative lter paper, ashless, Grade 41, Sigma-Aldrich). The crude extracts were frozen and freeze-dried (Christ ® LOC-1m, Germany) for 24 h to obtain a storable powder that could be used throughout the study. The reason for using water extract in this study was to mimic the scenario in the eld, where the saponin-rich plant products are often directly applied instead of the pure saponins. The saponins from three extracts were marked as quillaja sa- ponins (saponins from quillaja bark), tea saponins (saponins from tea seed coat powder) and quinoa saponins (saponins from quinoa seed coat powder), respectively. The reference saponin (Quil-A ® ) was a puri ed commercial Puri ed quillaja saponins, from Brenn- tag Biosector A/S, containing > 95% saponins, < 5% residue water, and it was marked as Quil-A in this study.

2.2. Organisms and acute toxicity tests methods

Twelve organisms were cultured and tested based on the ‘‘ OECD guideline for Testing of Chemicals ’’ or International Standard (ISO) , including plants, algae, worms, mollusks, arthropods, and microbes. An overview of the endpoints and test conditions used in the twelve tests is given in Table 1. The concentrations of tests were rstly set at 12.5, 25, 50, 100, and 200 mg/L (saponin extracts concentration). If the 50% effect was not reached with these concentrations, lower or higher con- centrations would be chosen in the adjusted tests. Apart from adjusting the concentration range, smaller intervals between con- centrations could be implemented as well in order to get a better-

described concentration-response curve in the cases where the slope was steep. Generally, the highest concentration used in the tests was 200 mg/L of saponins.

2.3. Semi-quanti cation of total saponin content of the extracts

To be able to compare the toxicity of the different plant extracts relative to their saponin content, a semi-quantitative method was used for determination of saponin. Quil-A was used as a standard, assuming 100% pure saponin, and relative saponin contents of quillaja extract, tea extract and quinoa extract were expressed as a weight percentage relative to Quil-A, w/w % (Quil-A/sample %). The method uses a vanillin-sulfuric acid assay and exploits the colour reaction of oxidized triterpene saponin backbones (agly- cones) with vanillin, which was measured as the absorbance at 480 nm using a UV/VIS spectrometer (Lambda 25, PerkinElmer) ( Cheok et al., 2014; Goel et al., 2012 ). For the vanillin-sulfuric acid assay, 100 mL samples were added to a 20 mL glass vial containing 0.3 ml of vanillin reagent (8% vanillin, w/v in 99.9% ethanol) and 3 ml of 72% (v/v) sulfuric acid placed in an ice water bath. The samples were then heated in a 60 C water bath for 10 min and cooled in the ice water bath before measuring absorbance. In order to reduce the effect of the matrix in the extract on the absorbance, standard addition was used as an analytical method ( Harris, 2010 ), which is further described in Supplementary Materials.

2.4. Statistics

Concentration-response curves of the 12 acute toxicity tests were performed and plotted using the open source software R, version 3.3.2 (R Team, 2014 ). Data was described with a three parameter log-logistic regression model using the drc-package ( Ritz and Streibig, 2005 ), assuming normal or binary distribution

Table 1 Experimental conditions of the individual toxicity tests and the guidelines followed. Climate chamber conditions are given as temperature ( C) and light:dark period in hours (L:D). Irradiance is given in mmol/m 2 /s Photosynthetic Active Radiation (PAR) for the photosynthesizing organisms.

Organisms

Taxonomic

Age/Size

Duration Endpoints

Medium

Climate chamber

Replicates per concentration

References

group

conditions

 

Vibrio scheri

Proteobacteria e

 

30 min Luminescence inhibition

2% NaCl

10 ± 1 C in dark

Three replicates

( ISO, 2007 )

Pseudokirchneriella

Chlorophyta e

48 h

Relative

growth

Algae growth

20

± 1 C, 24:0 h,

Three replicates

(

OECD,2006a )

subcapitata

rate

medium

90

Lemna minor

Tracheophyta Two

weeks old

7 d

Relative

growth

K-medium

24 ± 1 C,

16:8 h,

Six replicates with one frond each

( OECD, 2006b )

 

rate

100

 

Daphnia magna

Arthropoda

24 h

old

48 h

Immobilisation

M7

medium

20

±

1 C,

16:8 h

Four replicates with ve neonates in each

( OECD, 2004 )

Chironomus riparius Arthropoda

Fourth instar larvae Around 10 mm

48

h

Immobilisation

M7 medium

20 ± 1 C,

16:8 h

Four replicates with ve larvae ( OECD, 2011 ) in each Four replicates with ve larvae ( OECD, 2011 ) in each

Chaoborus

Arthropoda

48 h

Immobilisation

M7 medium

20

±

1 C,

16:8 h

crystallinus

Gammarus pulex

Arthropoda

Random size ( > 5 mm) Random size ( > 10 mm) Random size ( > 10 mm)

48

h

Mortality

Arti cial freshwater

10

± 1 C, 12:12 h Twelve replicates with one gammerus in each

(

Rasmussen

   

et al., 2016 )

Tubifex tubifex

Annelida

48

h

Mortality

Arti cial freshwater

20

± 1 C, in dark Four replicates with ve worms ( OECD, 2007 )

     

in each

Lumbriculus

Annelida

48

h

Mortality

Arti cial freshwater

20

±

1 C,

16:8 h

Four replicates with ve worms ( OECD, 2007 ) in each

variegatus

   

Lymnaea stagnalis

Mollusca

12 weeks old (5 e 10 mm)

48

h

Mortality

Arti cial freshwater Arti cial freshwater Arti cial freshwater

20

±

1 C,

16:8 h

Four replicates with ve snails ( Ducrot et al.,

     

in each

2014

)

Lymnaea stagnalis

Mollusca

24 h

old

7 d

Mortality

20

±

1 C,

16:8 h

Sixteen replicates with one embryo in each

( Hallett et al.,

embryo Danio rerio embryo Chordata

   

2016

)

24 h old

4 d

Mortality

26

±

1 C in dark

Five replicates with two embryos in each

( OECD, 2013 )

V. scheri was a BioTox kit with lyophilized bacteria, from Aboatox Oy, Finland. C. crystallinus and T. tubifex were bought from an aquarium store (Neon sken) in Copenhagen, Denmark. L. variegatus was originally from Department of Science and Environment, Roskilde University, Denmark. G. pulex was collected from a small stream in the north of Copenhagen, coordinates: 55 48 0 58 00 N 12 18 0 45 00 E (d m's ’’ ). L. stagnalis were originally from Department of Biology, University of Southern Denmark. All acquired organisms were acclimatized to growth conditions for at least two days prior to testing. D. rerio embryo was provided by Department of Veterinary Disease Biology, University of Copenhagen, Denmark. Other organisms were kept and cultured in our own lab. All used mediums such as K-medium, M7 medium and Arti cial freshwater are described in the previous papers ( Jessing et al., 2014; OECD, 2011; Rasmussen et al., 2016 ).

X. Jiang et al. / Environmental Pollution 236 (2018) 416e 424

419

of data depending on the endpoint monitored (Equation (1) ):

y ¼

1 þ d x b

e

(1)

where y is the measured endpoint (survival/growth rate/inhibited rate); d corresponds to the untreated controls; e is the concentra- tion causing 50% lethality or effect, depending on the endpoint measured; b is proportional to the slope around e , and x is the treatment concentration. E(L)C 50 (the concentrations causing 50% lethality/effect) was retrieved and used for the SSDs. SSDs are generated using a log- logistic distribution (Equation (1) ) ( Xu et al., 2015 ), using open source software R version 3.3.2 based on the drc-package. In the SSD model, y in Equation (1) is the proportion of affected species, x is the saponin concentration of different E(L)C 50 , and e is the saponin concentration of 50% species affected. The proportion of affected species was calculated as shown in Equation (2) ( Posthuma et al., 2002 ).

y i ¼

R

i

n þ

1

(2)

where y i is the proportion of affected species; R i is the rank of species and n is the number of E(L)C 50 values used in the model. From the SSDs, a hazardous concentration (HC p ) is identi ed at which a certain percentage (p) of all species is assumed to be affected. HC 5 is derived from the SSD model using the software R. Species not reaching and E(L)C 50 value at the highest saponin concentration tested (200 mg/L) were included in the rank but not in the SSD- t. The EFSA Aquatic Guidance Document suggests that a larger than toxicity value can be used in the SSD if this value is higher than the highest non-censored value ( EFSA PPR, 2013 ). In principle, the PEC of saponins should be estimated using implemented exposure scenarios and models (e.g. FOCUS). As little guidance to the application is given and the input required to model surface runoff and leaching is not available, we choose to use a simple worst-case loading scenario based on spray drift (Equa- tion (3) ) ( EFSA PPR, 2013 ). The biopesticide environmental con- centration, expressed as the mass that enters the water per surface area of water, can be obtained with knowledge of water depth, eld dose per area, and percent spray drift. In this case, we assume a eld dose on 30 cm depth water with 30% spray drift deposition.

c ¼ D sd App

h

(3)

where c is the saponin concentration in surface water (g/m 3 ); D sd is the spray drift deposition as fraction of the application dose; App is the application dose given as mass per surface area (h/m 2 ) and h is the surface water depth (m).

3. Results

3.1. Semi-quanti cation of total saponins

The total saponin content in the different plant extracts was estimated using a standard addition method, and the standard addition curves of quillaja extract, tea extract, quinoa extract are shown in Supplementary Materials, Fig. S1. The resulting total saponin contents of the quillaja, tea and quinoa extracts were estimated to be 67.2 ± 1.2%, 57.3 ± 1.3%, and 67.9 ± 1.4% of saponins in the dried extracts, respectively (w/w % Quil-A/sample %). The E(L)C 50 and HC 5 of the different saponin extracts shown in this study are expressed as total nominal saponin concentrations

calculated from the weight of the dried extracts dissolved in the stock solutions multiplied with the saponin percentage.

3.2. Acute toxicity results

The E(L)C 50 of the different saponin-rich plant extracts are shown in Table 2 . The concentration-response curves are shown in Supplementary Materials ( Figs. S2 e S13 ). The full concentration- response curves of the acute tests were conducted on quillaja extract and the reference saponin, Quil-A. Some organisms were not sensitive to saponins and 50% effect was not reached even at 200 mg/L of saponins. This was the case for Vibrio scheri, Chiro- nomus riparius, Chaoborus crystallinus and Gammarus pulex . Those organisms were considered as insensitive species, and the whole acute tests were therefore not conducted on tea extract and quinoa extract, but the highest concentration (200 mg/L of saponins) was tested to show no effect. Therefore, E(L)C 50 of those insensitive species is expressed as above 200 mg/L. To the reference Quil-A, there is more than 20-fold difference in toxicity between the most sensitive and most insensitive species, with the E(L)C 50 estimates varying from 11.9 mg/L to more than 200 mg/L. The most sensitive species was the Danio rerio ( sh) embryos. A similar pattern was shown for quillaja saponins, the crude extract from the same plant species. Tea saponins showed higher selectivity between species, with an approximately 100-fold difference in species sensitivity, varying from the lowest LC 50 of ~2 mg/L for the sh embryo, followed by the snail, L. stagnalis , the two worm species Tubifex tubifex and Lumbriculus variegatus to over 200 mg/L for the arthropod C. crystallinus . Quinoa saponins seem to be the least toxic of the three plant extracts, when given on a relative saponin content basis, with a concentration of 49.5 mg/L for the snail embryo test being the lowest LC 50 value.

3.3. Species sensitivity distributions

The SSDs are constructed using the E(L)C 50 values in Table 2 and are shown in Fig. 2 . The data of Lymnaea stagnalis is not included in the SSD approaches since the more sensitive life-stage ( L. stagnalis embryo) is used. In addition, in order to make all dataset compa- rable and consistent, the data of L. stagnalis is also not included in tea saponin SSD approach, even though the snail adult is more sensitive than the embryo in this case. Therefore, there are 11 E(L) C 50 values applied to construct the SSD giving n ¼ 11 for Equation (2) . The species that has an E(L)C 50 value > 200 mg/L are considered in the rank, but data are not included in the SSD- t. Detailed regression parameters of SSDs are shown in Table 3 . Generally, all species display the same overall pattern in terms of sensitivity. The most sensitive species are worms, sh and snail embryos, while the tested microbe and arthropods are relatively insensitive. Quillaja saponins and the standard saponin Quil-A, have similar SSD curves ( Fig. 2 A, D), which was expected as they originate from the same plant species. Due to the higher variance in species sensitivity towards tea extracts, the tea saponins SSD curve is relatively shallow with a b parameter of e 0.58 ( Fig. 2 B). Hence, the retrieved HC 5 -value is almost 10-times lower than the LC 50 of the most sensitive species and the coef cient of variation (CV) of the parameter gets relatively high (50%, as opposed to the 34, 26 and 30% for quillaja saponins, quinoa saponins and Quil-A, respec- tively). The quinoa saponins are the least toxic and the SSD curve is relatively steep ( 1.21) according to the PPP Regulation, de ning steep as there is less than 100-fold difference between the least and most sensitive species ( EFSA PPR, 2013 ) ( Fig. 2 C). The differences in the species sensitivity towards the different plant extracts has the consequence that the saponin concentration derived from quinoa saponins protecting 95% of the species (to a 50% effect level) could

420

X. Jiang et al. / Environmental Pollution 236 (2018) 416e 424

Table 2 E(L)C 50 of saponin-rich plant extracts towards different organisms. All values are given ± standard error with 95% con dence limits around E(L)C 50 given in brackets.

Organisms

E(L)C 50 (mg/L) towards different organisms

 

Quillaja saponins

Tea saponins

Quinoa saponins

Quil-A

Vibrio scheri Pseudokirchneriella subcapitata

> 200

> 200

>

200

>

200

146 ± 18.7 (109, 183) 103 ± 11.7 (80.1, 126) 18.3 ± 1.53 (15.3, 21.3) 313 ± 79.5 (157, 469)

63.4

± 3.91

>

200

>

200

 

(55.7, 71.1)

 

Lemna minor

60.8

± 4.68

>

200

92.8 ± 11.9

(51.6, 70.0)

(69.5, 116)

Daphnia magna

15.1

± 2.49

190

± 60.4

22.9

± 0.73

(10.2, 20.0) 104 ± 17.2 (70.3, 138)

(71.6, 308)

(21.5, 24.3) 108 ± 8.17 (92.0, 124)

Chironomus riparius

283

± 25.3

(233, 333)

Chaoborus crystallinus

>

200

> 200

>

200

> 200

Gammarus pulex

>

200

148 ± 79.2 (0, 303)

>

200

115 ± 32.0 (52.3, 178)

Tubifex tubifex

47.6 ± 3.74 (40.3, 54.9) 40.1 ± 4.01 (32.2, 48.0) 52.1 ± 96.5 (0, 241) 37.0 ± 3.76 (29.6, 44.4) 9.02 ± 1.30 (6.47, 11.6)

2.77

± 0.25

127

± 11.5

37.3

± 2.18

(2.28, 3.26)

(105, 150)

(33.0, 41.6)

Lumbriculus variegatus

1.95

± 0.16

85.8

± 8.63

39.0

± 40.5

(1.64, 2.26)

(68.9, 103)

(0, 118)

Lymnaea

stagnalis

2.23

± 0.36

65.6

± 113

31.5

± 28.3

 

(1.52, 2.94)

(0, 287)

(0, 87.0)

Lymnaea stagnalis embryo

10.7

± 1.35

49.5

± 7.12

18.7

± 2.81

(8.05, 13.3)

(35.5, 63.5)

(13.2, 24.2)

Danio rerio embryo

1.93

± 0.27

89.6

± 13.6

11.9

± 1.80

(1.40, 2.46)

(62.9, 116)

(8.37, 15.4)

All concentrations expressed in saponin concentration relative to Quil-A.

affect nearly 50% of the species if they had been derived from tea saponins, as the HC 5 -value of quinoa saponins is close to HC 50 - value of tea saponins ( Table 3 ). The median HC 5 derived from the SSD model, giving the highest concentration protecting 95% of species to a 50% effect level, are shown in Table 3 . There is approximately 100-fold difference be- tween HC 5 -values of tea saponins and quinoa saponins, which are the most and least toxic saponins in this study. However, there is only around 8-fold difference between tea saponins and quinoa saponins when comparing the HC 50 -values. This demonstrates clearly the effect the slope of the SSD has on HC 5 -values, as also mentioned above. The HC 5 -values were used to calculate quality criteria for surface waters according to the WFD and PPP Regulation, as MAC-EQS and RAC sw,ac , respectively ( Table 4 ). MAC-EQS is derived from median HC 5 -values divided by an assessment factor of 10, and RAC sw,ac using an assessment factor between 3 and 6. Here we use the higher value of 6, as the factor between the lowest and highest E(L) C 50 values used to construct the SSD curve is less than 100 ( EFSA PPR, 2013 ). For tea saponins, it could, however, be discussed if the assessment factor should be lower, due to the relatively low slope of the curve and the consequent conservative HC 5 -value ( Fig. 2 B). To get an estimate of potential surface water concentrations, we used Equation (3) assuming a eld dose with 30% spray drift deposition on 30 cm depth water and a 10% saponin content in the solid product (10% saponins content is estimated by U.S. Environmental Protection Agency, 2007 ) and no degradation or sorption ( EFSA PPR, 2013 ). As of cial recommended doses are rarely given, we used doses given in the literature together with what they were used for and the references. The PECs in the surface water of quillaja saponins, tea saponins and quinoa saponins are given in Table 4 .

4. Discussion

The present study con rms earlier observations of soft skin organisms, such as worms, snails and sh embryos, being the most sensitive to saponins. This is believed to be due to saponins detergent-like structures, causing damages to cell membranes by creating pores, vesiculation and membrane domain disruption,

ultimately killing the organisms ( Augustin et al., 2012 ). Surpris- ingly, the microbe, V. scheri, is extremely insensitive to all three kinds of saponins. This is unexpected since saponins are supposed to have a high cytotoxicity to single-cell organisms, because of their membrane disrupting properties, and because of their documented antibacterial activities ( Dinda et al., 2010 ). Study on rumen mi- crobes, however, has shown 100% degradation of saponins within 24 h ( Makkar and Becker, 1997 ). Hence, some microbes may be resistant to the soap-like properties of saponins and might rather use them as substrates. This possibility cannot be excluded to occur for V. scheri . Moreover, a recent study shows an EC 50 of quillaja saponin towards V. scheri of 578 mg/L, supporting our V. scheri test has an EC 50 > 200 mg/L ( De Oliveira et al., 2017 ). The study also shows that organisms in their early stage are more sensitive to saponins than more grown individuals. This is evident for L. stagnalis where the embryo is more vulnerable than 5 e10 mm grown individuals for three of the four extracts. The similarity in which species are the most sensitive across the different saponin- rich extracts illustrates that the saponins most likely have a similar mode of action. The activity of the different saponin extracts may depend both on the composition of the saponins and on the non-saponin con- tent of the extracts, such as tannins and polyphenol. Comparing Quil-A and quillaja saponins, however, they have very similar SSD patterns and similar HC 5 -values ( Fig. 2 , Table 3 ). This suggests that it is the saponins that contribute most to the bioactivity, at least in this species, while the non-saponin content contributes less. For the much more toxic tea saponins, we do, however not know if other components do also contribute to toxicity. Studies of the molluscicidal activity of tea extract and tea saponins do, however, show similar responses indicating that the saponins in the tea extract are the main toxic compounds (LC 50 of tea saponin is 0.66 mg/L, while it is 6.79 mg/L for the extract) ( Kijprayoon et al., 2014 ). The much lower toxicity of saponins from quinoa saponins compared to the standard quillaja saponins (Quil-A) emphasizes that the difference in the structure and composition of the saponins from different plant species does affect their bioactivity and resulting toxicity. We, therefore, recommend not using surrogate or read-across data during registration of saponin-rich plant extracts

X. Jiang et al. / Environmental Pollution 236 (2018) 416e 424

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et al. / Environmental Pollution 236 (2018) 416 e 424 421 Fig. 2. Species sensitivity distribution

Fig. 2. Species sensitivity distribution curves. A : quillaja saponins, B : tea saponins, C: quinoa saponins, D : reference quillaja saponins (Quil-A). All concentrations are expressed in total saponin concentrations. The dots are input data, E(L)C 50 values of different species, while the line it the regression line based on Equation (2) .

Table 3 Summary of regression parameters of the SSD model for quillaja saponins, tea saponins, quinoa saponins and reference quillaja saponins (Quil-A), gi ven together with the HC 5 of different saponins. All values are given ± standard error with 95% con dence limits around E(L)C 50 given in brackets.

Parameters

Stressors

Quillaja saponins

Tea saponins

Quinoa saponins

Quil-A

b

(proportional to the slope around e )

0.82 ± 0.10 (-1.02, 0.62) 105 ± 11.0 (83.4, 127) 2.91 ± 1.00 (0.95, 4.87)

0.58 ± 0.06

1.21 ± 0.20 (-1.60, 0.82) 260 ± 25.7 (210, 310) 22.9 ± 5.84 (11.5, 34.4)

1.06 ± 0.12

 

(-0.70, 0.46)

(-1.30, 0.82)

e

(HC 50 , mg/L)

34.4

± 5.17

72.0

± 6.93

 

(24.3, 44.5)

(58.4, 85.6)

HC 5 (mg/L)

0.22

± 0.11

4.42

± 1.33

(0, 0.44)

(1.81, 7.03)

for the use as pesticides or for other purposes, due to the strikingly different bioactivities. Assuming that only the aglycone is the causative agent of bioactivity, neglecting the effect of the sugar moiety, we may

attempt to make an overview of bioactivity of the different sapo- nins ( Fig. 3 ). There are many types of aglycones in saponins depending on the plant species it derives from, and often the functional groups differ from each other as well. Although quillaja

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X. Jiang et al. / Environmental Pollution 236 (2018) 416e 424

Table 4 Predicted environmental concentration (PEC) in the aquatic environment based on doses given in the literature together with the water quality crite ria MAC-EQS and RAC sw,ac . All concentrations are given in mg/L.

Saponin-rich

Objectives

Dosing

PEC a MAC- RAC sw,ac References EQS

plant products

Liquid quillaja extract Tea seed coat

For control/suppression of pathogenic fungi or plant parasitic nematodes in vegetable or fruit elds Expel earthworms in turfgrass

1.4 e 3.7 g liquid/m 2 30.0 g powder/

m

2

0.1

0.29

0.49

( U.S. Environmental Protection

e

0.4

Agency, 2007 )

3

0.02

0.04

( Potter et al., 2010 )

   

Quinoa seed

For control/suppression of pathogenic fungi or bacteria in vegetable elds and 0.6 e 2.0 g

0.1

2.29

3.82

( Joshi et al., 2008; U.S. Environmental

coat

Golden apple snail ( Pomacea canaliculat a) in rice water

coat/m 2

e

0.2

Protection Agency, 2005 )

dose

g

m

30% 10%

a Assuming a eld dose with 30% spray drift deposition on 30 cm depth water and a 10% saponin content in the solid product, which is PEC ¼

2

.

30 cm

content in the solid product, which is PEC ¼ 2 . 30 cm Fig. 3. Structures

Fig. 3. Structures of different aglycones depending on the functional groups attached to the R 1 and R 2 positions.

saponins, tea saponins and quinoa saponins may include many types of aglycones, the most abundant ones being reported for the three species are shown in Fig. 3 ( Güçlü-Üstünda g and Mazza, 2007). There are basically three types of very abundant aglycone structures present, which are de ned by their functional groups at the C-4 position. Quillaic acid has an aldehyde ( e CHO) group, hederaginin has a hydroxymethyl ( e CH 2 OH) group and oleanolic acid has a hydrogen atom ( e H) at the C-4 position ( Fig. 3 ). While aglycones of quillaja saponins mainly consist of quillaic acid, tea saponins mainly consist of quillaic acid and hederagenin ( Kitagawa et al., 1998 ), while quinoa saponins contain approximately 50% of hederagenin and 25% of oleanolic acid while rest are unknown aglycones ( Ruiz et al., 2017 ). As we observe tea saponins to be up to 10-fold more toxic to some organisms than quillaja saponins, it suggests that the oleanolic acid type of aglycone may be relatively inactive. Hence, the additional functional groups at C-4 of quillaic acid and hederagenin seem to be essential to the bioactivity, no matter if it is an aldehyde or a hydroxymethyl group. A study of saponins from Barbarea vulgaris which reported that the type of saponins with oleanolic acid had a less antifeedant effect on her- bivorous insects than the ones with hederagenin supports this

observation of the importance of the aglycone structure ( Augustin et al., 2012 ). Other studies show that the functional group of aldehyde at C-4 is essential for quillaja saponin bioactivities (anti- body stimulation and induction of an immune response), as de- rivatives modi ed at this function (without aldehyde group) did not show adjuvant activity ( Moses et al., 2014 ). Despite this, there are studies showing that aldehyde is a more important functional group for adjuvant effects than the other two groups ( e CH 2 OH, e H) ( Greatrex et al., 2015 ), as oleanolic acid has a higher cytotoxic ac- tivity when measured on cancer and normal cells than quillaic acid and hederagenin ( Barthomeuf et al., 2002; Gauthier et al., 2009; Podolak et al., 2010 ). Hence, the relative bioactivity of aglycones may differ depending on the endpoint measured. The effects of the sugar moieties on bioactivity can, however, not be ignored. Numerous studies con rm that the bioactivity of sa- ponins is in uenced both by the aglycone and the sugar moieties ( Podolak et al., 2010 ), especially the sugar attached at the C-3 po- sition. The study on Barbarea vulgaris saponins reports that ole- anolic acid and hederagenin with a sugar chain attached in C-3 position increased the antifeedant effect, compared to those that have no sugar attached ( Augustin et al., 2012 ). Pilot studies in our

X. Jiang et al. / Environmental Pollution 236 (2018) 416e 424

423

own lab have shown a large decrease in toxicity of both quillaja saponins and tea saponins towards daphnids, when cleaving off all sugar chains (only aglycone left) (Unpublished data). However, the sugar moieties are extremely different and complex among quillaja saponins, tea saponins and quinoa saponins, hence, more re- searches are needed to explain the effect of different sugar chains on different types of bioactivity. The difference in bioactivity between the saponins of different plant origin was also re ected in the tentative risk assessment done on the plant extracts. The SSD-based water quality criteria MAC- EQS and RAC sw, ac exceeded the worst case predicted environ- mental concentration 100-fold in the case of tea saponins, while the quality criteria were not exceeded for quinoa saponins and were on the borderline of being so for quillaja saponins ( Table 4 ). Hence, saponin-rich plant extracts may pose a risk, if not used cautiously. More knowledge on use and fate in the environment is, however, needed to be able to do a proper risk assessment. Likewise, analytical techniques to measure saponins in the environment would be an advantage, though this may be a big challenge due to the structural diversity of plant-produced saponins.

Acknowledgements

We wish to thank Assoc. Prof. Sven-Erik Jacobsen, University of Copenhagen, for providing Quinoa seed coat powder, NOR-ADD A/ S, Denmark, for providing tea seed powder, Assoc. Prof. Henrik Holbech, University of Southern Denmark, for providing the culture of Lymnaea stagnalis , Assoc. Prof. Annemette Palmqvist, University of Roskilde, for providing the culture of Lumbriculus variegatus , and Assist. Prof. Louise von Gersdorff Jørgensen, University of Copen- hagen, for providing Danio rerio embryos. We also want to give our gratitude to the nancial support of University of Copenhagen and China Scholarship Council.

Appendix A. Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.envpol.2018.01.058 .

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