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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:
From January 30th to March 16th, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
adopted on 23 March 2006
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Tested concentrations were determined at the beginning and at the end of the test period. During the range finding study and main study, analytical samples (plus retain samples) were taken from a randomly chosen replicate per treatment group, at beginning and at the end of the test (i.e. at 0 hour and 7 days) in order to determine the concentration of test item during the test.
About 10 ml of sample was collected from all test concentration in duplicates.
Vehicle:
no
Details on test solutions:
Required quantity of test item was taken in a beaker and small volume of Swedish Standard (SIS) growth medium was added and stirred well using glass rod. After complete suspension, test formulation was transferred to measuring cylinder, beaker was rinsed with Swedish Standard (SIS) growth medium and transferred again to measuring cylinder; the rinsing procedure was repeated until the complete transfer of test contents. Finally, Swedish Standard (SIS) growth medium was added up to required volume.
A homogeneous distribution of the test item within the stock solution was achieved by stirring on magnetic stirrer before preparation of the test medium.
Test organisms (species):
Lemna minor
Details on test organisms:
TEST ORGANISM
- Source: in house maintained culture of Lemna minor, obtained from the Department of Botany, Tumkur University was used as the test system for the study.
- Pre-culture: from healthy axenic culture of Lemna minor, pre-culture was prepared of desired colonies to provide the inoculum for test cultures.

BREEDING CONDITIONS
- Subculturing: Lemna minor culture is periodically subcultured.
- Illumination: 6500 to 10000 lux
- Temperature: 22 to 26 °C.
- Medium: Lemna minor was maintained separately on Steinberg medium and Swedish Standard (SIS) media.

PRECULTURE
- Test vessels: 10 beakers containing 100 ml of culture medium.
- Duration: preculture was initiated 7 days before start of the test.
- Incubation conditions: pre cultures are incubated under same conditions as the test cultures.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
24.2 - 25.5 °C
pH:
Test vessels: 6.43 - 6.88
Control: 6.52 - 6.56
Nominal and measured concentrations:
3.7, 7.7, 16.2, 34.0, 71.4 and 150.0 mg/l, nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: sterilized, glass beakers of 150 ml capacity.
- Type of cover: the test vessels were covered with perforated transparent sheet to minimize accidental contamination, while allowing necessary air exchange.
- No. of fronds per colony: the initial Lemna minor colonies comprising 3 fronds; each test replicates contain 12 fronds during the main study.
- No. of vessels per concentration: 6 replicates.
- No. of vessels per control: 6 replicates.

GROWTH MEDIUM
- Standard medium used: yes; Swedish Standard (SIS). Stock solution was prepared freshly during every renewal period in the Swedish Standard (SIS) growth medium

OTHER TEST CONDITIONS
- Light intensity and quality: 6640 to 6984 lux during range finding study and the main study.

EFFECT PARAMETERS MEASURED
- Counting of frond number: the frond number at the start on day 0, day 4 and on day 7 were counted in each test vessel and recorded.
- Observation of fronds: frond numbers appearing normal or abnormal were determined at beginning of test and on day 3, day 6 and at end of the test. Changes in plant development were recorded.
- Dry weight: dry weight was determined at test start from representative sample of the inoculum culture. Consistent with the approach taken in the test. At the end of the test, the plant material from each replicate of test concentration and control vessel was used to determine dry weight. To determine the dry weight they are blotted to remove excess water and then dried at 60 °C to a constant weight. Any root fragments were included and dry weight was measured.

ENVIRONMENTAL PARAMETERS
The temperature (culture medium) and light intensity (at points the same distance from the light source as the Lemna fronds) was monitored once in a day during pre-culture and exposure during range finding study and limit test. The pH of the test concentrations (fresh and spent) were measured on day 0 and on day 7 at the termination of the test during range finding and limit test.

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l.
- No. of fronds per colony: the initial Lemna minor colonies comprising 3 fronds; each test replicates contain 9 fronds for exposure during range finding study.
- Replicates: 2 replicates in each concentration and 4 replicates in control.
- Results used to determine the conditions for the definitive study: the % inhibition in growth rate for frond number was found to be 2.2, 3.3, 4.9, 15.1, 29.8 and 72.1 % at the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l. The % inhibition in yield for frond number was found to be 5.4, 7.8, 11.1, 30.7, 52.7 and 88.6 % at the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l respectively.

VALIDITY CRITERIA OF THE TEST
The Lemna growth inhibition test fulfills the validity criteria of the OECD guideline 221.
The doubling time of frond number in the control was 2.364 days (56.7 hours) and 2.423 days (58.2 hours), corresponding to average specific growth rate of 0.293 and 0.286 d-1 during range finding study and the main study, respectively (validity criterion: growth rate ≥ 0.275 d-1, doubling time <2.5 d).
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
47.94 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Probit analysis
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
26.04 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Remarks on result:
other: Probit analysis
Details on results:
MAIN TEST
The NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) for frond number were found to be 3.7 and 7.7 mg/l, respectively.
The NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) for dry weight were found to be 16.2 and 34.0 mg/l, respectively.

Counting and observation of frond number
The initial number of frond number in all treatment replicates on day 0 was 12. The mean frond numbers on day 7 was 89.0 in control and 86.7, 74.7, 61.3, 49.7, 38.3 and 15.0 in the vessels with test item concentrations of 3.7, 7.7, 16.2, 34.0, 71.4 and 150 mg/l.
All fronds appeared normal on day 0, 3, 6 and on day 7 in control and at the tested concentrations of 3.7 and 7.7 mg/l. Some fronds appeared chlorosis on day 3, 6 and 7 at the tested concentrations of 16.2, 34.0 and 71.4 mg/l. Fronds appeared chlorosis on day 3, 6 and 7 in the tested concentration of 150 mg/l.

Dry weight
On day 0 dry weight was found to be 0.0233 g. At the end of the test, the mean dry weight of each group was found to be 0.125 g in control and 0.122, 0.109, 0.106, 0.071, 0.055 and 0.017 g were observed in the vessels with test item concentration of 3.7, 7.7, 16.2, 34.0, 71.4 and 150 mg/l.

DOSE-RANGE FINDING
Counting and observation of frond number
On day 7, the mean frond numbers per group was 70.3 in control and 67.0, 65.5, 51.5, 38.0 and 16.0 in the tested vessels at the concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100 mg/l.
All fronds appeared normal on day 0, 3, 6 and on day 7 in control and at the test concentrations of 0.01, 0.1 and 1.0 mg/l. Fronds appeared normal until day 3, while some fronds appeared chlorosis on day 6 and 7 in the vessels with test item concentrations of 10.0, 50.0 and 100.0 mg/l.

Dry weight
On day 0 dry weight was found to be 0.0195 g. At the end of the test, the mean dry weight was found to be 0.234 g in control and 0.219, 0.166, 0.137, 0.092, 0.056 and 0.042 g in the vessels with test item concentration of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l.

MEASURED CONCENTRATIONS
During the range finding and the main study, samples from the test concentration was analyzed on day 0 and on day 7 and the results reported that test samples are found to be with in the acceptable range ± 20 % recovery to the nominal concentration.
Results with reference substance (positive control):
The ErC50 values for growth rate of frond number and dry weight were calculated to be 3.66 and 3.26 mg/l, respectively.
The ErC50 value for the frond number of 3.66 is in good agreement with the results of an international ring test between 2.7 and 3.4 mg/l (n=52).

INHIBITION IN GROWTH RATE AND YIELD DURING MAIN STUDY

Test conc. (mg/l) % Inhibition in growth rate % Inhibition in yield
Frond number Dry weight Frond number Dry weight
0 0 0 0 0
3.7 1.3 0.6 3.0 2.3

7.7

8.8

5.6

18.6

12.5

16.2

18.6

7.1

36.0

15.4

34.0

29.1

24.1

51.0

43.0

71.4

42.2

34.4

65.8

56.0

150.0

88.9

70.1

96.1

86.4

INHIBITION IN GROWTH RATE AND YIELD DURING RANGE FINDING STUDY

Test conc. (mg/l)

% Inhibition in growth rate

% Inhibition in yield
Frond number Dry weight Frond number Dry weight
0 0 0 0 0
0.01 2.2 2.0 5.4

6.1

0.1

3.3

11.8

7.8

28.9

1.0

4.9

18.5

11.1

41.4

10

15.1

31.9

30.7

56.5

50

29.8

47.1

52.7

76.5

100

72.1

54.8

88.6

81.8

MEASURED CONCENTRATIONS

MAIN STUDY

Nominal concentration (mg/l)

Day 0

Day 7

Concentration (mg/l)

% Recovery

Concentration (mg/l)

% Recovery

0

-

-

-

-

3.7

3.84

103.78

3.75

101.35

7.7

7.68

99.74

7.48

97.14

16.2

15.49

95.62

15.0

92.56

34.0

37.22

109.47

35.34

103.94

71.4

74.28

104.04

70.67

98.98

150.0

149.99

99.99

140.63

93.75

RANGE FINDING STUDY

Nominal Concentration (mg/l)

Day 0

Day 7

Concentration (mg/l)

% Recovery

Concentration (mg/l) % Recovery
0 - - - -
0.01 - - - -
0.1 - - - -
1 1.02 101.5 1.09 109
10 10.2 101.95 10.23 102.3
50 50.88 101.76 51.06 102.12
100 103.87 103.87 101.67 101.67
Validity criteria fulfilled:
yes
Remarks:
doubling time of frond number in the control was 2.364 days and 2.423 days, corresponding to average specific growth rate of 0.293 and 0.286 d-1 during range finding study and main study, respectively.
Conclusions:
ErC50 (7d): 47.94 mg/l, based on frond number
EyC50 (7d): 26.04 mg/l, based on frond number
Executive summary:

The test item was assessed for Lemna sp. as per OECD guideline 221.

Lemna minor was exposed to the test item, under static condition, over a period of 7 days.

Swedish Standard (SIS) growth medium was used. Based in the results of range finding study, the main study was performed at the test concentration of 3.7, 7.7, 16.2, 34.0, 71.4 and 150.0 mg/l. Lemna minor were observed for frond number on day 0, 4 and on day 7. Observation for fronds was made at beginning of test and on day 3, 6 and at end of the test day 7.

During the main study, all fronds appeared normal on day 0, 3, 6 and on day 7 in control and in tested concentrations of 3.7 and 7.7 mg/l. Some fronds appeared chlorosis on day 3, 6 and 7 in the tested concentrations of 16.2, 34.0 and 71.4 mg/l. Fronds appeared chlorosis on day 3, 6 and 7 in the tested concentration of 150 mg/l.

The ErC50 and EyC50 values for growth rate and yield for frond number were found to be 47.94 and 26.04 mg/l, respectively, with 95 % confidence limits for growth rate of 30.35 and 75.75 mg/l and 17.99 and 37.68 mg/l 95 % confidence limits for yield.

The ErC50 and EyC50 values for growth rate and yield for dry weight was found to be 91.63 and 45.66 mg/l, respectively; with 95 % confidence limits for growth rate of 74.54 and 112.64 mg/l and 95 % confidence limits for yield of 38.82 and 53.70 mg/l.

The NOEC and LOEC for frond number were found to be 3.7 and 7.7 mg/l, respectively. The NOEC and LOEC for dry weight was found to be 16.2 and 34.0 mg/l, respectively.

Conclusion

ErC50 (7d): 47.94 mg/l, based on frond number

EyC50 (7d): 26.04 mg/l, based on frond number

Description of key information

ErC50 (7d): 47.94 mg/l, based on frond number

EyC50 (7d): 26.04 mg/l, based on frond number

Key value for chemical safety assessment

EC50 for freshwater plants:
47.94 mg/L
EC10 or NOEC for freshwater plants:
3.7 mg/L

Additional information

Test item was assessed on Lemna sp., as per OECD guideline 221.

Lemna minor was exposed to test item, under static condition, over a period of 7 days. Swedish Standard (SIS) growth medium was used. Based in the results of range finding study, the main study was performed at the test concentration of 3.7, 7.7, 16.2, 34.0, 71.4 and 150.0 mg/l.

During the main study, all fronds appeared normal on day 0, 3, 6 and on day 7 in control and in tested concentrations of 3.7 and 7.7 mg/l. Some fronds appeared chlorosis on day 3, 6 and 7 in the tested concentrations of 16.2, 34.0 and 71.4 mg/l. Fronds appeared chlorosis on day 3, 6 and 7 in the tested concentration of 150 mg/.

The ErC50 and EYC50 values for growth rate and yield for frond number were found to be 47.94 and 26.04 mg/l, respectively, with 95 % confidence limits for growth rate of 30.35 and 75.75 mg/l and 17.99 and 37.68 mg/l 95 % confidence limits for yield.

The ErC50 and EYC50 values for growth rate and yield for dry weight was found to be 91.63 and 45.66 mg/l, respectively; with 95 % confidence limits for growth rate of 74.54 and 112.64 mg/l and 95 % confidence limits for yield of 38.82 and 53.70 mg/l.

The NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) for frond number were found to be 3.7 and 7.7 mg/l, respectively. The NOEC and LOEC for dry weight was found to be 16.2 and 34.0 mg/l, respectively.

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