Supplementary material for Jessing et al. “Potential Ecological Roles of Artemisinin
Produced by Artemisia annua L.”
Materials and Methods
Enchraeus crypticus toxicity test The toxicity of artemisinin to the survival and reproduction of the soil-dwelling annelid E. crypticus w as determined using a protocol modified from the (OECD, 2004) for testing of chemical effects on enchytraeids. This reproduction test has been validated and standardized (Rombke and Moser, 2002). The enchytraeids used were reared from cultures originally established from worms obtained by the terrestrial ecotoxicology group at Vrije Univeristeit, Amsterdam. The worm culture from the Amsterdam stock was placed in incubators at 18°C under constant light. The worms were fed weekly with a mixture of autoclaved oat grains and fish food (protein content 33.5%). The soil used was the LUFA 2.2 standard soil. This medium is a loamy sand soil with a pH of 5.5

1.1, organic carbon content of 2.1

0.4, cation exchange capacity of 10

0.5 meq/100g, and water holding capacity of 55%.
For each concentration, four replicates per treatment and six per controls were prepared. Each replicate consisted of
10 g dry weight of LUFA soils spiked with a stock solution of artemisinin in acetone. Concentrations used were 7.8,
15.6, 31.3, 62.5, 125, 250, and 500 mg/kg soil. After spiking, the soils were left in the fume hood overnight to let the acetone evaporate. The soils then were moistened to 50% of water holding capacity (27.5% moisture content) using demineralized water. Five adult worms were added to the soil surface of each replicate. Worms were fed with autoclaved oat grains and fish food and the containers were closed to prevent water loss. The containers were placed in a constant temperature facility at 18 o C and incubated under constant light. Once a week the beakers were weighed, and demineralized water was added to the wells to compensate for evaporation loss of water, 25 mg of ground oats with fish food were added, and molding foods were removed. All treatments were harvested after 4 weeks, when visible off-spring was present in the controls. Five ml of ethanol was added to each beaker to fix the enchytraeids and after 1 minute 300 µl 1% Bengal red was added and mixed carefully. The beakers were left at 5 ° C overnight for the worms to stain. The next day as much of the content of the well as possible was moved to a plastic container with a small spoon. Each container was then washed well 2-3 times with 10 ml of distilled water. The soil slurry was sieved through a coarse (1 mm) and fine (0.18 mm) mesh to extract the stained worms, and adults and offspring were then counted on a white phototray under magnification.
Caenorhabditis elegans toxicity test. The nematode Caenorhabditis elegans of the N2 Bristol strain (obtained from the C. elegans Genetics Centre) were cultivated at 18±0.5
o C in darkness on plates of a modified bacteriological agar
(nematode growth medium (Brenner 1974)) and fed E. coli , of the uracil deficient strain OP50. The cultures were maintained by transferring a chunk of agar from the existing culture plates to freshly prepared nematode growth medium agar plates each week.

Toxicity tests were performed in three types of media: on agar plates, in soil and in water. The artemisinin concentrations tested in the experiment in which the worms lived on agar plates were 0, 0.2, 0.46, 0.91, 1.82, 3.66,
7.31, 14.63, 29.25, and 58.5 mg artemisinin/L agar. Stock solutions of artemisinin were prepared in acetone, and controls with and without acetone were included. The agar was spiked before agar hardening when it was approximately 60 o C. All experiments were conducted using age-synchronized nematodes that were exposed to artemisinin throughout their full life cycle. For synchronization, adult egg-producing worms were picked from the culture and placed on control or artemisinin-dosed agar. They were left at 18 o C for 4 h and allowed to lay eggs.
After this time, the adults were removed and the eggs laid were left to hatch. The offspring (test organisms) were then grown under the relevant test conditions on these batch plates until they reached the L4 larval stage, which took approximately 3 d. Thereafter, 12 nematodes for each artemisinin treatment were transferred to artemisinin-spiked agar in 12-well plates seeded with E. coli as a source of food. In all cases, one individual was placed in each well.
The treatments were arranged in a random design. Thereafter, all test plates were kept in the dark at 18 o C. During the test, the nematodes were moved to new wells every 24 h, and their individual daily offspring were counted through the reproductive stage and senescence until the test organisms died. Fertile eggs and hatched juveniles were counted as offspring, while unfertilized oocytes were excluded. Individuals that disappeared or were found dead on the side of the wells were excluded from the results.
The toxicity test in soil was a 4-d test performed according to Hoss et al. (2009). LUFA 2.2 standard soil (0.3 g) soil dried at room temperature and passed through a 5 mm mesh was placed in each well of five 12-well plates. Four plates with two replicates per treatment were used. For the controls, the soil was moistened with 0.2 ml nematode medium (Brenner 1974) per well and mixed with 0.5 ml of the uracil deficient strain OP50 suspension. Artemisinin stock solution was prepared in acetone and diluted with nematode medium before mixing into the soil and 0.5 mL
OP50 suspension was added. Soil concentrations of 1.56, 7.8, 39, 195, and 975 mg artemisinin/kg soil were tested.
The plates were left in a fume hood for the acetone to evaporate. Five L1 worms were then transferred to each well, and the plates were placed at 18ºC. Examples of the remaining worms were photographed under stereomicroscope for determination of size. After 24 h, one plate was heat killed at 50ºC and 0.5 ml of rose bengal solution was added to each well for coloration of the worms. The same procedure was repeated after 48, 72, and 96 h. To count the worms, soil and worms were removed from the wells by adding approximately 5 ml Ludox
®
colloidal silica suspension (TM50 mixed with deionized water to a density of 1.13 g/ml) to the well and removing the suspension with a Pasteur pipette to a 15-ml centrifuge tube. The suspension was mixed on a vortex mixer and centrifuged at
800 g for 10 min. The supernatant was purred into a Petri dish, the pellet was re-suspended with Ludox suspension and the procedure repeated. The nematodes which were retained in the viscous Ludox suspension were counted under a stereomicroscope at 25-fold magnification. The mother worms were removed and photographed for size determination.

The toxicity test in water with C. elegans were conducted with one adult worm per well for 96 h, to monitor total reproduction or with five L1 worms per well for 96 h to monitor growth and initial reproduction. The ISO guideline for pore water was used (ISO, 2010). Artemisinin stock solution was prepared in acetone. Dilutions were made in
OP50 stock solution. The artemisinin concentrations tested were 3.175, 6.25, 12.5, 25, and 50 mg/L. Controls with and without acetone were included. One ml test solution was added to each well in 10 12-well plates in a randomized system and left in the fume hood for the acetone to evaporate. A 0.5 ml OP50 suspension (1000 FAU) was added to each well. Then, either one adult or 5 L1 larvae were added per well and incubated at 20 o C for 96 h.
After 96 h, 1 ml rose bengal solution was added per well, and the worms were killed by heating to 60 o C for 20 minutes. The samples were stored at 5 o C until counting and measurements of worms using a stereomicroscope.
Daphnia magna toxicity tests.
A Daphnia magna acute immobilization test was carried out according to the OECD
Guideline for Testing of Chemicals (OECD, 2004). For each dose, 20 neonates <24 h old were tested. For controls,
40 neonates in clean media and another 40 neonates in media with the maximal solvent concentration were used.
Concentrations were made in aerated M7 medium. Tested concentrations were 0.0485, 0.097, 0.195, 0.39, 0.78,
1.56, 3.125, 6.25, 12.5, 25, and 50 mg artemisinin /L. Daphnia were divided into groups of 5, each placed in 25 mL media in a 100-mL glass beaker. Immobility, abnormalities and changes in the behavior of D. magna were observed visually and registered after 24 and 48 h. Dissolved oxygen and pH were measured in the highest test concentrations and in the controls in the beginning and at the end of the tests (Hach HQ40d, Hach, Loveland, USA). Neither of the parameters changed significantly during the experiments with the pH average being 7.65 ± 0.09 and the oxygen saturation 96.6 ± 2.2 % at 21°C. The sensitivity of the culture and culture conditions were tested with the reference substance potassium dichromate (K
2
Cr
2
O
2)
. EC50 values were in average 1.05 ± 0.25 mg/L, which is within the range of 0.6 – 2.1 mg/L prescribed by the guideline (OECD 2004).
Supplementary material results and discussion
Enchraeus crypticus . There was no observed effect of artemisinin on E. crypticus in the tested range of soil concentrations (7.8-500 mg/kg).
Caenorhabditis elegans. There was no observed effect of artemisinin on either growth or reproduction of C. elegans in the tested range of soil (1.56-975 mg/kg) and water (3.175-50 mg/L) concentrations. However, the test on agar had a significant response of reproductive output to artemisinin (Fig. 1). The parameters obtained by modeling with the three parameter log-logistic dose-response model were EC
50
= 12.4±7.2 mg/L, d = 46.6±5.9 and b = 0.75±0.26.
It is curious that the agar test showed a decreasing effect on reproduction within a test-range where no effects were observed in the soil and water test. The effect concentrations are, however, in the mg/L range, which are levels far too high for artemisinin to have a notable effect within realistic concentration ranges in soil (Jessing et al, 2009,
Hermann et al, 2012).

60
50
40
30
20
10
0
1 10 100
Artemisinin (mg L-1)
Fig. 1 Fig. 2: Number of eggs and juveniles of C. elegans after 4 d of exposure to artemisinin on agar plates. Data are given as mean ± Standard error ( N = 12, for control N = 22). The three parameter log-logistic dose response model fitted to data is shown as a solid line.
Daphnia magna . The mobility of D. magna was not affected by artemisinin at concentrations up to the solubility limit of the compound (Fig. 2). The tendency was, however, that artemisinin inhibited mobility slightly at the highest concentration, both after 24 and 48 h. Data could, nonetheless, not be described by a concentration response model. Hence, contrary to the sensitivity of two other tested aquatic organisms, the algae Pseudokirchneriella subcapitata and the macrophyte Lemna minor having EC
50
values of 0.24
± 0.01 mg/L and 0.19
± 0.03 mg/L (Jessing et al, 2009), the aquatic crustacean D. magna was very insensitive to artemisinin.
0.8
0.6
0.4
1.2
1.0
0.2
0.01
Control 0.1
1
Artemisinin (mg L
-1
)
10 100
Fig. 2 Mobile D. magna divided by total no. as a function of artemisinin concentration after 24 (black circles) and
48 (open circles) h. Data are given as mean ± standard error ( N = 4, for control N = 12).

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