Registration Dossier

Ecotoxicological information

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Jan 2017 to 18 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state / Appearance: Solid / red
- Total Organic Carbon: 612 mg/g
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: ambient temperature, in a tightly sealed container without exposure to light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Since the test item reflects a reaction mass of components with varying water solubilities, the main component concentrations were measured for the purpose of testing the stability of the test item. In the definitive test, samples were collected from all test item loading rates and the control for chemical determination at exposure initiation, at renewals and termination.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Appropriate amounts of the test item were weighed separately in glass flasks with 20X AAP medium and mixed with appropriate volume of the 20X AAP medium. The mixtures of a loading of 100, 40, 16, 6.4 and 2.56 mg/L were visually heterogeneous (with visible undissolved particles) and colored. The mixtures and the control (20X AAP medium) were left for mechanical shaking for 48 hours (30ºC, 90 rpm, darkness). After 48 hours of mechanical shaking, the mixtures (visually heterogeneous) and the control were left at room temperature for a settling phase (no shaking). After 24 hours of settling, the mixtures were visually heterogeneous. All mixtures and the control were filtrated, first through a conditioned paper filter (qualitative cellulose filter) and next through a conditioned nitrocellulose membrane filter (filter, 0.22 Um, Millipore). Filtration was chosen as the separation method since centrifugation was not possible with the large volumes of test solution. Each filtrate was visually homogeneous (without any visible undissolved particles). From each filtrate and the control, two samples were collected for chemical determinations. One was transferred to chemical analyses at exposure initiation, others were stored under test conditions.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: duckweed
- Strain: CPCC 310
- Original stock source: Stock G3 from Canadian Phycological Culture Centre (CPCC), Department of Biology, University of Waterloo, Ontario, Canada. The test culture was further cultivated by the test laboratorium.

CULTURING
Duckweed Lemna gibba was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacity of 600 mL with transparent lids and incubated at room temperature with constant illumination (pre-culturing). The duckweed culture was inoculated to fresh medium once a week. A pre-culture was started nine days before exposure. Only organisms in good physiological condition without any discolouration were used for the inoculation of the test item concentrations and the control.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 h

Test conditions

Hardness:
304.1 mg/L (as CaCO3)
Test temperature:
24.3 - 24.5 °C
pH:
- Test start: 7.61 - 7.71
- Test end: 8.55 - 8.94
Nominal and measured concentrations:
- Nominal loading rates: 0 (control), 2.56, 6.4, 16, 40 and 100 mg/L
- Measured concentrations: The substance was found to be stable in the test solutions (80 - 120% of nominal, see 'Any other information on materials and methods incl. tables')
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass crystallisers, with a depth of 4 cm and a diameter of 7 cm.
- Type: Closed with transparent lids to minimise evaporation and accidental contamination, allowing necessary air exchange.
- Fill volume: 150 mL
- Renewal rate of test solution: Two renewals (at Day 2 and 4)
- No. of colonies per vessel: 3
- No. of fronds per colony: 3
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes; 20X AAP
- Aeration: 24h
- Adjustment of pH: yes, when necessary with 0.1M HCl
- Alkalinity: 304.9 mg/L as NaHCO3

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: Temperature was measured continuously. The light intensity was measured at exposure initiation, twice during exposure and at exposure termination with a 2π receptor lux-meter (Sonopan L-100, Poland). pH was determined in each fresh test item loading rate and the econtrol at exposure initiation and at each renewal. The pH values were measured in each old test item loading rate and the control at each renewal and at exposure termination.

OTHER TEST CONDITIONS
- Light intensity: Mean light intensity of 7180 - 7350 lux (approximately 94 – 99 µE·m-2·s-1); constant illumination.
- Light source: Fluorescent light source (cool white light, Philips Master PL-L 24W/840/4P / Philips Master TL5 HO 24W/840/1 SL/40)

EFFECT PARAMETERS MEASURED
- Determination of frond number: on day 2, 4 and at exposure termination. Only visible distinct fronds were counted.
- Biomass: The dry weight of the representative sample of the duckweed culture used as the inoculum was measured at exposure initiation. The dry weight of all plants from each test vessel was measured after exposure termination. All colonies (with roots) were transferred onto previously weighed microscopic slides and dried at approximately 60°C in a laboratory oven until constant weight
- Other: At the same time, observations of plant development were performed: frond size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony break-up or loss of buoyancy, root length and appearance.

RANGE-FINDING STUDY
- Test concentrations: 100-fold diluted filtrate of a loading of 10 mg/L, 10-fold diluted filtrate of a loading of 10 mg/L, filtrate of a loading of 10 mg/L and filtrate of a loading of 100 mg/L.
- Results used to determine the conditions for the definitive study: The growth rate inhibition based on the frond number was 1.9% in the 100-fold diluted filtrate of a loading of 10 mg/L, 11.7% in the 10-fold diluted filtrate of a loading of 10 mg/L, 30.6% in the filtrate of a loading of 10 mg/L and 80.1% in the filtrate of a loading of 100 mg/L. The growth rate inhibition based on the dry weight was 1.4% in the 100-fold diluted filtrate of a loading of 10 mg/L, 8.4% in the 10-fold diluted filtrate of a loading of 10 mg/L, 26.4% in the filtrate of a loading of 10 mg/L and 79.3% in the filtrate of a loading of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
28.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
frond number
Remarks on result:
other: 95% C.L.: 27.49 – 29.97 mg/L
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
3.48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
frond number
Remarks on result:
other: 95% C.L.: 3.15 - 3.82 mg/L
Details on results:
No specific effects were observed at any test concentration, unless stated here: On day 2, in the test item filtrates of a loading of 100 and 40 mg/L, coloured fronds were observed. On day 4, in the test item filtrate of a loading of 16 mg/L, coloured fronds were observed. In the test item filtrates of a loading of 100 and 40 mg/L, the colony broke up and spots of chlorosis or necrosis were observed. At exposure termination, in the test item filtrate of a loading of 6.4 mg/L, slightly coloured fronds were observed. In the test item filtrates of a loading of 100, 40 and 16 mg/L, the colony broke up and separated roots and spots of chlorosis or necrosis were observed. Inhibition of growth rate based on frond number was observed from the lowest test concentration (10.8%) and increased in a dose-dependent manner up to 76.2% inhibition at a loading rate of 100 mg/L. See 'Any other information on results incl. tables'.
Results with reference substance (positive control):
The 7-d ErC50 based on frond number was determined to be 9.94 mg/L (95% C.L.: 8.14 - 12.12). The 7-d ErC10 was determined to be 4.71 mg/L (95% C.L.: 1.10 - 6.48).
Reported statistics and error estimates:
Probit method calculations and analysis by Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity (with Residuals), Williams multiple sequential t-test procedure, Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm.

Any other information on results incl. tables

Table: Inhibition of growth rate and yield

Filtrate of a loading of

Based on frond number

Based on dry weight

% inhibition at exposure termination (day 7) (growth rate)

% inhibition at

exposure

termination (day 7)

% inhibition at

exposure

termination (day 7)

% inhibition at

exposure

termination (day 7)

Control

0

0

0

0

2.56 mg/L

10.8

30.9

4.5

14.3

6.4 mg/L

15.5

41.4

10.6

30.2

16 mg/L

34.7

70.5

35.9

71.3

40 mg/L

61

89.8

58.1

88.1

100 mg/L

76.2

95.3

80.3

96.2

Table: Endpoint values based on growth rate

Endpoint

Frond number

Dry weight

0-2 d

0-4 d

0-7 d

0-7 d

ErL10

16.85

(13.22 – 20.28)

5.8

(5.25 – 6.36)

3.48

(3.15 – 3.82)

4.94

(4.30 – 5.59)

ErL20

30.37

(26.01 – 34.41)

11.99

(11.19 – 12.79)

7.18

(6.69 – 7.67)

9.12

(8.24 – 10.00)

ErL50

93.78

(84.04 – 106.88)

48.02

(45.90 – 50.31)

28.7

(27.49 – 29.97)

29.48

(27.74 – 31.35)

LOELR

40

≤ 2.56

≤ 2.56

≤ 2.56

NOELR

16

< 2.56

< 2.56

< 2.56

Table: Endpoint values based on yield

Endpoint

Frond number

Dry weight

0-2 d

0-4 d

0-7 d

0-7 d

EyL10

11.19

(8.90 – 13.43)

2.76

(2.50 – 3.03)

< 2.56

< 2.56

EyL20

19.98

(17.07 – 22.75)

5.28

(4.91 – 5.65)

< 2.56

3.89

(3.60 – 4.18)

EyL50

60.60

(55.14 – 67.14)

18.23

(17.45 – 19.05)

7.03

(6.73 – 7.34)

9.83

(9.38 – 10.30)

LOELR

40

≤ 2.56

≤ 2.56

≤ 2.56

NOELR

16

< 2.56

< 2.56

< 2.56

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'
Conclusions:
The test substance is acutely harmful for aquatic plants.