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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October from 20th to 23rd, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
No chemical analysis of the test concentrations was performed, assuming the test item to be stable in medium over the whole test period.
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
adopted 23th March 2006
Deviations:
yes
Remarks:
not affecting the study outcomes
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on test solutions:
At all concentrations, the test item was fully dissolved, no suspended matter of supernatant was visible. The colour of the test solutions were light blue, dark blue and almost black at the concentrations 1, 10 and 1000 mg/l respectively.
Test organisms (species):
Lemna minor
Details on test organisms:
TEST ORGANISM
- Source: Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Ab-wasserentsorgung, Schichauweg 58, D-12307 Berlin.
- Method of cultivation: at least seven days before testing, sufficient colonies are transferred aseptically into fresh sterile medium and cultured for 7 - 10 days under the conditions of the test.
- Pre-culture: exponential growing plant monoculture of Lemna minor.

CONDITIONS
- Illumination: continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China).
- Temperature: 24 ± 2 °C
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
The temperature ranged between 22.0 and 25.5°C during the whole test period.
pH:
Blank control: 5.6 at the start and 6.4 at the end.
Test vessels: 5.7 at the start and 4.9-6.5 at the end.
The pH values did mot drift by more than 1.5 units during the test.
Nominal and measured concentrations:
10, 100 and 1000 mg/l, nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 400 ml beakers, all-glass, with 200 ml of test medium.
- Type of cover: the beakers are covered with black paper up until the 200 ml mark to ensure that illumination comes only from above and not from the sides.
- Starting frond number: 9-12 fronds per replicate, 2-4 fronds per plant.
- No. of vessels per concentration: test medium, test item and duckweed, three replicates per concentration.
- No. of vessels per control: test medium and duckweed, six replicates.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM
- Preparation of dilution water: recommended STEINBERG medium, prepared with ultra-pure water.
- Macroelements:
KNO3: 350 mg/l
KH2PO4: 90 mg/l
K2HPO4 x 3H2O: 16.5 mg/l
MgSO4 x 7H2O: 100 mg/l
Ca(NO3)2 x 4H2O: 295 mg/l
- Microelements
H3BO3: 120.0 µg/l
ZnSO4 x 7H2O: 180.0 µg/l
Na2MoO4 x 2H2O: 44.0 µg/l
MnCl2 x 4H2O: 180.0 µg/l
FeCl3 x 6H2O: 760.0 µg/l
C10H14N2Na2O8 x 2H2O: 1500.0 µg/l

OTHER TEST CONDITIONS
- Adjustment of pH: the pH of the STEINBERG medium was adjusted before the test (pH 5.5 ± 0.2).
- Light intensity and quality: continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Light-ing F18T8/6500K EUP (CH Lighting CO., Ltd., China); homogeneity ±15 %.
- Incubation: beakers are incubated on a black non-reflecting surface.

EFFECT PARAMETERS MEASURED
The number of fronds were counted on days 0, 3, 5 and 7.
The dry weight (duckweed dried at 60 °C overnight) was determined at day 0 in six additional blank replicates (similar to those used in the test), and on day 7 in all blanks and all test vessels.
Changes in plant development, e.g. in frond size, appear-ance, indication of necrosis, chlorosis or gibbosity, colony break-up, loss of buoyancy, root length and appearance were noted.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol, yearly testing laboratory check
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks:
respect to growth rate and to yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
respect to growth rate and to yield
Details on results:
EFFECTS ON FROND NUMBER
With respect to growth rate inhibition for the endpoint frond number, the following effects as compared to the untreated controls were observed: 15 % at 1000 mg/l. No significant effects were observed at 100 and 10 mg/l, as determined by Williams’ test.
With respect to yield inhibition for the endpoint frond number, the following effects as compared to the untreated controls were observed: 31 % at 1000 mg/l. No significant effects were observed at any of the three concentrations, as determined by Williams’ test.
The no-observed-effect concentrations of the test item to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint frond number were both 100 mg/l nominal concentration (active ingredient), as determined by Williams’ test.

EFFECT ON DRY WEIGHT
With respect to growth rate inhibition for the endpoint dry weight, no significant effects were observed at any of the three concentrations, as determined by Williams’ test. With respect to yield inhibition for the endpoint dry weight, no significant effects were observed at any of the three concentrations, as determined by Williams’ test.
The no-observed-effect concentrations of test item to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint dry weight were both 1000 mg/l nominal concentration (active ingredient), as determined by Williams’ test.

APPEARANCE OF THE TEST AND CONTROL PLANTS AT THE END OF THE TEST
In the control the plants were healthy, as well as at 10 mg/l. At 100 mg/l, the plants were agglomer-ated. At 1000 mg/l, the plants were partially colored in bleu by the dye (which is a sign of chlorosis) and were agglomerated.
Results with reference substance (positive control):
According to guideline ISO/CD 20079 “Determination of the toxic effect of water constituents and waste water to duckweed (Lemna minor) - Duckweed growth inhibition test”, the ErC50 value based on the frond number should lie in the range 1.8–3.6 mg/l; this level of sensitivity was attained (data from 9th March 2015).

Frond number between days 0 and 7 and the corresponding growth rate inhibition

Nominal conc. (mg/l) of the active ingredient Replicate Frond N., data Frond N., growth rate
d0 d3 d5 d7 GR* µ7-0/d Doubling time (d)** Inhibition (%) Inhibition mean value (%)
Control A 12 27 35 79 0.27 2.57 8.6 0.0
B 12 32 51 113 0.32 2.16 -8.6
C 12 29 40 94 0.29 2.36 0.2
D 12 27 44 93 0.29 2.37 0.7
E 12 28 35 74 0.26 2.67 11.8
F 12 30 51 122 0.33 2.09 -12.5
10 A 12 32 42 113 0.32 2.16 -8.8 -10.1
B 12 29 56 136 0.35 2.00 -17.7
C 12 29 49 102 0.31 2.27 -3.8
100 A 12 30 37 77 0.27 2.61 9.8 2.6
B 12 29 43 87 0.28 2.45 3.9
C 12 33 57 107 0.31 2.22 -6.1
1000 A 12 32 41 68 0.25 2.80 15.9 14.8
B 12 20 35 66 0.24 2.85 17.3
C 12 37 48 75 0.26 2.65 11.1

*average specific growth rate over 7 days

**average doubling time of controls must be < 2.5d; here, doubling time was 2.4 days.

Frond number between days 0 and 7 and the corresponding yield inhibition

Nominal conc. (mg/l) of the active ingredient Replicate Frond N., data Frond N., yield
d0 d3 d5 d7 Yield days 0-7 Inhibition (%) Inhibition mean value (%)
Control A 12 27 35 79 67 20.1 0.0
B 12 32 51 113 101 -20.5
C 12 29 40 94 82 2.2
D 12 27 44 93 81 3.4
E 12 28 35 74 62 26.0
F 12 30 51 122 110 -31.2
10 A 12 32 42 113 101 -20.5 -25.2
B 12 29 56 136 124 -47.9
C 12 29 49 102 90 -7.4
100 A 12 30 37 77 65 22.5 6.6
B 12 29 43 87 75 10.5
C 12 33 57 107 95 -13.3
1000 A 12 32 41 68 56 33.2 31.2
B 12 20 35 66 54 35.6
C 12 37 48 75 63 24.9

Dry weight at days 0 and 7 and the corresponding growth rate inhibition

Nominal conc. (mg/l) of the active ingredient Replicate Dry weight, data Dry weight, growth rate
d0* (mg) d7 (mg) GR** µ7-0/d Inhibition (%) Inhibition mean value (%)
Control A 2.2 9.5 0.21 12.7 0.0
B 2.2 16.0 0.28 -18.4
C 2.2 11.3 0.23 2.4
D 2.2 10.6 0.22 6.2
E 2.2 8.6 0.19 18.7
F 2.2 16.9 0.29 -21.6
10 A 2.2 16.5 0.29 -20.2 -19.8
B 2.2 16.8 0.29 -21.3
C 2.2 15.9 0.28 -18.0
100 A 2.2 8.1 0.19 22.2 4.5
B 2.2 11.1 0.23 3.4
C 2.2 14.4 0.27 -12.1
1000 A 2.2 10.5 0.22 6.8 9.4
B 2.2 8.8 0.20 17.3
C 2.2 11.0 0.23 4.0

*average of six frond samples similar to those used in the test

**average specific growth rate over 7 days.

Dry weight at days 0 and 7 and the corresponding yield inhibition

Nominal conc. (mg/l) of the active ingredient Replicate Dry weight, data Dry weight, yield
d0* (mg) d7 (mg) Yield days 0-7 Inhibition (%) Inhibition mean value (%)
Control A 2.2 9.5 7.30 26.6 0.0
B 2.2 16.0 13.80 -387.0
C 2.2 11.3 9.10 8.5
D 2.2 10.6 8.40 15.6
E 2.2 8.6 6.40 35.7
F 2.2 16.9 14.70 -47.7
10 A 2.2 16.5 14.30 -43.7 -42.7
B 2.2 16.8 14.60 -46.7
C 2.2 15.9 13.70 -37.7
100 A 2.2 8.1 5.90 40.7 9.5
B 2.2 11.1 8.90 10.6
C 2.2 14.4 12.20 -22.6
1000 A 2.2 10.5 8.30 16.6 20.6
B 2.2 8.8 6.60 33.7
C 2.2 11.0 8.80 11.6

*average of six frond samples similar to those used in the test

PHYSICO-CHEMICAL OBSERVATIONS

The pH-values (measured in the pooled replicates) at the beginning of the test were 5.6 in the blank control and 5.7 in all test concentrations. At the end of the test, pH-values were 6.4 for the blank control, 6.5 at 10 mg/l, 5.0 at 100 mg/l and 4.9 at 1000 mg/l. The pH values did mot drift by more than 1.5 units during the test.

The light intensity was 6090–8140 lux (mean 6668 lux; max. variation ± 22 %) at the start of the test and 6760–7920 lux (mean 7718 lux, max. variation ± 11 %). The light homogeneity was not in the 15% range, but since the test vessels were randomly re-placed and since the growth criterion in the control was fulfilled, this deviation does not have a significant impact on the outcome of the study.

The temperature ranged between 22.0 and 25.5 °C during the whole test period; which is within the required 24 ± 2 °C.

At all concentrations, the test item was fully dissolved, no suspended matter of supernatant was visible. The colour of the test solutions were light blue, dark blue and almost black at the concentrations 1, 10 and 1000 mg/l respectively.

Validity criteria fulfilled:
yes
Remarks:
doubling time of frond number in the control was less than 2.5 days (60 h), corresponding to approximately a 7-fold increase in 7 days and an average specific growth rate of 0.275 d-1
Conclusions:
Based on frond number, ErC50 and EyC50 (7d) > 1000 mg/l (nominal)
Based on dry weight, ErC50 and EyC50 (7d) > 1000 mg/l (nominal)
Executive summary:

The inhibitory effects of the test item to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and dry weight, according to the OECD guideline 221. Since the test item is soluble, the nominal concentrations were prepared by dilution of a stock solution of the test item in STEINBERG medium. The test was performed at nominal concentrations of 10.9, 109 and 1090 mg/l of test item, corresponding to 10, 100 and 1000 mg/l of the active ingredient. No chemical analysis of the test concentrations was conducted. The determination of the effect concentrations was based on the nominal concentrations of the active ingredient assuming the test item to be stable in medium for the whole test period. At all concentrations, the test item was fully dissolved, no suspended matter of supernatant was visible. The colour of the test solutions were light blue, dark blue and almost black at the concentrations 1, 10 and 1000 mg/l respectively.

Considering the frond number and with respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 15 % at 1000 mg/l. No significant effects were observed at 100 and 10 mg/l, as determined by Williams’ test. With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 31 % at 1000 mg/l. No significant effects were observed at 100 and 10 mg/l, as deter-mined by Williams’ test.

Considering the dry weight and with respect to growth rate and yield inhibition, no significant effects were observed at any of the three concentrations, as determined by Williams’ test.

Conclusion

Based on frond number, ErC50 and EyC50 (7d) > 1000 mg/l (nominal)

Based on dry weight, ErC50 and EyC50 (7d) > 1000 mg/l (nominal)

Description of key information

Based on frond number, ErC50 and EyC50 (7d) > 1000 mg/l (nominal)

Based on dry weight, ErC50 and EyC50 (7d) > 1000 mg/l (nominal)

Key value for chemical safety assessment

EC50 for freshwater plants:
1 000 mg/L
EC10 or NOEC for freshwater plants:
100 mg/L

Additional information

The inhibitory effects of the test item to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and dry weight. The nominal concentrations were prepared by dilution of a stock solution of the test item in STEINBERG medium. The test was performed at nominal concentrations of 10.9, 109 and 1090 mg/l of test item, corresponding to 10, 100 and 1000 mg/l of the active ingredient. No chemical analysis of the test concentrations was conducted. At all concentrations, the test item was fully dissolved, no suspended matter of supernatant was visible. The colour of the test solutions were light blue, dark blue and almost black at the concentrations 1, 10 and 1000 mg/l respectively.

Considering the frond number and with respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 15 % at 1000 mg/l. No significant effects were observed at 100 and 10 mg/l, as determined by Williams’ test. With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 31 % at 1000 mg/l. No significant effects were observed at 100 and 10 mg/l, as determined by Williams’ test. Considering the dry weight and with respect to growth rate and yield inhibition, no significant effects were observed at any of the three concentrations, as determined by Williams’ test.